WO2003066652A2 - Jlp cytoplasmic scaffolding protein and nucleic acids encoding it - Google Patents

Abstract

A cytoplasmic scaffolding protein named JLP has been isolated, which tethers MEKK3, MKK4, JNK,p38 MAPK, C-Myc and MAX into a sign aling module that controls the apoptotic response. Nucleic acid sequences which encode JLP, and methods of using the JLP nucleic acids and proteins, and mutants thereof, to modulate the apoptotic response in cells are provided.

Description

JLP CYTOPLASMIC SCAFFOLDING PROTEIN AND NUCLEIC ACIDS ENCODING THE SAME

Reference to Government Grant

The invention described herein was supported in part by the National

Institute of Health, under grant nos. CA09214 and GM49897. The U.S. government has certain rights in this invention.

Field of the Invention

The invention relates to novel cytoplasmic scaffolding proteins, nucleic acid sequences which encode the proteins, and methods of using the nucleic acids, proteins and mutants thereof to modulate the apoptotic response in cells.

Background of the Invention

The mitogen-activated protein (MAP) kinases comprise a family of kinases responsive to a variety of environmental stimuli. Generally, the MAP kinases are activated in response to an extracellular signal, and in turn activate other molecules (e.g., transcription factors) which affect cellular functions through a series of phosphorylations. In this way, the MAP kinases play a critical role in relaying external signals into the cell nucleus.

The MAP kinases can be divided into three subgroups; extracellular signal-regulated kinase (ERK); c-Jun NH2-terminal kinase (JNK); and p38 MAP kinase (Brunet and Pouyssegur (1997), Essays Biochem. 32: 1-16; Cano and Mahadevan (1995), Trends. Biochem. Sci. 20, 117-22; Davis (1995), Mol. Reprod. Dev. 42: 459-67; Dhanasekaran and Reddy (1998), Oncogene 17: 1447- 55; English et al. (1999), Exp. Cell. Res. 253: 255-70; Minden and Karin (1997), Biochim. Biophys. Ada. 1333: F85-104; Kyriakis and Avruch (1996), Bioessays 18: 567-77; Nebreda and Porras (2000), Trends Biochem. Sci. 25: 257-60). ERK is mainly activated by proliferative signals, while JNK and p38 MAPK are activated by environmental stress signals, such as pro-inflammatory cytokines, UN light, ionizing radiation, chemotoxic drugs, heat stress, oxidative stress and osmotic stress (Kyriakis and Avruch (1996), supra; Nebreda and Porras (2000), supra; Obata et al. (2000), Crit. Care. Med. 28: N67-77; Raingeaud et al. (1995), J. Biol. Chem. 270: 7420-6).

MAP kinase-mediated cell signaling events can affect cell growth or promote apoptotic responses, depending on the nature of the stimuli and/or the combination of the signaling pathways that are activated (Dhanasekaran and Reddy (1998) supra). For example, INK and p38 MAP kinase activations are associated with cell death in one system, and cell survival and growth in another (Jacobs-Helber et al. (2000), Blood 96: 933-40; Roulston et al. (1998), J. Biol. Chem. 2T___: 10232-9). It is also known that MAP kinase phosphorylation cascades can activate transcription factors such as Fos, Jun and Myc, although the precise mechanisms through which these transcription factors are recruited are not clear (Brunet and Pouyssegur (1997) supra; Dhanasekaran and Reddy (199%) supra).

INK and p38 MAP kinases are usually redundant in function. However, in certain systems co-activation of JNK and p38 MAP kinase can result in a different response than activation of either one alone.

For example, activation of either JNK or p38 MAP kinase alone leads to hypertrophy in cardiomyocytes, whereas co-activation of JNK and p38 MAP kinase in these cells leads to vacuolization and cell death, with suppression of hypertrophic responses (Wang et al. (1998), J. Biol. Chem. 273: 5423-6; Wang et al. (1998), J. Biol. Chem. 273: 2161-8). In contrast, erythropoietin-mediated survival of erythroid cell line HCD57 appears be mediated by the co-activation of JNK and p38 MAP kinase (Jacobs-Helber et al. (2000) supra).

Tumor necrosis factor (TNF) was found to induce JNK and p38 MAP kinase activity with biphasic kinetics (Roulston et al. (1998) supra). The first phase is transient and appears to result in cell survival, whereas the second phase appears to mediate apoptosis.

These observations suggest that the activity resulting from the co- activation of JNK and p38 MAP kinases is likely dependent on the nature of the transcription factors activated by these MAP kinases in response to extracellular signals. Thus, knowledge of the transcription factors and other components associated with JNK/p38 MAP kinase signaling pathways is critical to understanding how these pathways mediate the apoptotic response.

One transcription factor involved in JNK cell signaling pathway is c- Myc. c-Myc is a substrate of JNK (Noguchi et al. (1999), J. Biol. Chem. 274: 32580-7) and is phosphorylated by JNK at its Ser-62. Mutation of this serine residue results in the reduction of the transcriptional and transformational activities of c-Myc (Gupta et al. (1993), Proc. Natl. Acad. Sci. USA 90: 3216- 20; Pulverer et al. (1994), Oncogene 9: 59-70; Seth et al. (1991), J. Biol. Chem. 266: 23521-4). It is also known that activation of the JNK pathway results in increased transcriptional activity of c-Myc (Johnson et al. (1996), J. Biol. Chem. 271: 3229-37). Since the JNK and ρ38 MAPK signaling cascades are activated in response to environmental stresses such as pro-inflammatory cytokines, it is believed that c-Myc likely has a role in regulating the expression of genes involved in the environmental stress response. Other molecules involved in the JNK/p38 MAP kinase signaling pathways are the dual specificity MAP kinase kinases (MKKs); JNK is activated by MKK4, whereas p38 MAPK is activated by MKK3 and MKK4.

Cell-signaling via MAP kinases is facilitated by specialized molecules called scaffolding proteins, such as Ste5 in yeast (Choi et al. (1994), Cell 78: 499-512; Marcus et al. (1994), Proc. Natl. Acad. Sci. USA 91: 7762-6; Posas and Saito (1997), Science 276: 1702-5; Yablonski and Levitzki (1996), Proc. Natl. Acad. Sci. USA 93: 13864-9) and JSAP and JIP1-3 in mammalian cells (Elion (1998), Science 281: 1625-6; Kelkar et al. (2000), Mol. Cell. Biol. 20: 1030-43; Schaeffer et al. (1998), Science 281: 1668-71; Yasuda et al. (1999), Mol. Cell. Biol. 19: 7245-7254; Ito et al. (1999), Mol. Cell. Biol. 19: 7539-48; Ito et al. (2000), Gene 255: 229-234). Scaffolding proteins draw the various kinases of a signaling pathway - e.g., MEK kinases (MEKKs); MAP kinase kinases (MKKs) and MAP kinases - into close proximity and allow successive phosphorylation events in the signal cascade to occur efficiently. Moreover, by assembling a particular combination of kinases for activation, scaffolding proteins ensure specificity of a given signaling pathway. Scaffolding proteins also associate with other signal pathway components, such as transcription factors or other effectors of cellular functions.

A scaffolding protein and its associated kinases/other molecules is called a

"signaling module." One example of a signaling module is the Ste5/stel 1/Ste7/Fus3-Kssl module found in yeast.

Different combinations of kinases and transcription factors (and perhaps other molecule types) in a signaling module produce different effects in the cell upon activation by an external signal. A particular kinase or transcription factor can participate in more than one signaling module, but a given signaling module appears to contain a unique complement of molecules. Clustering of signal pathway molecules on a scaffolding protein insulates these molecules from the general cytosolic milieu, and decreases 'cross-talk' to other pathways during a signaling event. Moreover, signaling modules also allow greater signal amplification and speed than could be achieved with signal pathway molecules freely distributed in the cytosol. Signaling modules are therefore a form of spatial compartmentalization employed by the cell to ensure specificity and efficiency of a given signal transduction pathway.

Thus, the isolation and characterization of scaffolding proteins, and the proteins with which they interact, is a necessary step in understanding and controlling the biological effect of a signaling pathway. In particular, isolation and characterization of scaffolding proteins involved in JNK and p38 MAP kinase-mediated cell signaling is necessary for understanding and modulating the apoptotic response in cells. Modulating the apoptotic response in cells is desirable, if one wishes to selectively cause cell death or induce cell survival under a defined set of circumstances.

For example, it is common to treat subjects who have metastatic tumors with high doses of ionizing radiation. The radiation treatment, designed to destroy the tumor cells, can also destroy the subject's radiation-sensitive normal hematopoietic cells by inducing apoptosis. To protect these cells, a portion of the subject's bone marrow containing the hematopoietic stem cells can be removed prior to radiation therapy. Once the subject has been treated, the autologous hematopoietic stem cells are returned to the body. However, the initial radiation treatment is often followed by further doses of ionizing radiation, chemotherapy, or both. These further treatments may also kill the subject's normal hematopoietic stem cells. It is often not feasible to remove and bank the subject's bone marrow each time a radiation or chemotherapy treatment is required. Thus, it is desirable to prevent the death of hematopoietic stem cells from apoptosis-inducing environmental stresses, such as ionizing radiation or chemotherapy, experienced during treatment of cancer. Prevention of apoptosis can be achieved through control of the JNK/p38 signaling pathway. Thus, there is a need for isolation and characterization of the scaffolding protein which brings together the components of the JNKp38 MAP kinase signaling pathway; namely, MKK4, MKK3, MKK4, JNK, p38 MAP kinase, c- Myc and Max. Characterization of such a scaffolding protein would allow the modulation (i.e., induction or prevention) of the apoptotic response. Desirably, one could use the scaffolding protein to foster survival of certain cells under environmental stress conditions that would normally induce apoptosis, such as radiation or chemotherapy treatment for metastatic cancer.

Summary of the Invention

A scaffolding protein named "JLP" has been isolated, which tethers MEKK3, MKK4, JNK, ρ38 MAPK, c-Myc and MAX into a signaling module which controls the apoptotic response. JLP functions as a signaling conduit to transmit extracellular signals to the nucleus through MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module.

The present invention provides isolated mouse and human cDNA sequences, and fragments and homologs thereof, encoding the respective human and mouse JLP. Thus in one embodiment, the present invention provides an isolated nucleic acid sequence comprising SEQ ID NO: 1 (human JLP cDNA) or SEQ ID NO: 3 (mouse JLP cDNA), and fragments and homologs thereof. The invention also provides isolated nucleic acid sequences complementary to SEQ ID NOS: 1 and 3, and fragments and homologs thereof. The present invention also provides an isolated JLP encoded by SEQ ID

NO: 1 or SEQ ID NO: 3, and biologically active fragments, derivatives, homologs or analogs thereof. In one embodiment, JLP comprises the human

JLP of SEQ ID NO: 2. In another embodiment, JLP comprises the mouse JLP ofSEQ IDNO:4.

The invention also provides antibodies that bind to specific epitopes on human and mouse JLP, and to specific epitopes on derivatives, homologs, analogs, or antigenic fragments of JLP. The antibodies may be a monoclonal or polyclonal antibody, or an antibody fragment that is capable of specifically binding antigen.

The invention also provides a hybridoma that produces a monoclonal antibody which specifically binds the compounds of the invention.

The invention also provides a method of regulating the apoptotic response in cells, comprising the steps of contacting the cells with an effective amount of a compound comprising JLP or biologically active derivative, homolog or analog thereof, such that the compound is introduced into the cell and said modulation is effected. In a preferred embodiment, the modulation of the apoptotic response comprises inducing cell survival.

In a further embodiment, the invention provides a method of modulating the apoptotic response in cells by contacting a cell exposed to, or in danger of being exposed to, an apoptosis-inducing environmental stress with an effective amount of a compound comprising JLP or biologically active fragment, derivative, homolog or analog thereof, such that the cells do not undergo apoptosis in response to the environmental stress. In a still further embodiment, the invention provides a method of protecting hematopoietic stem cells from apoptotic death induced by anti-cancer radiation or chemotherapy treatments, comprising the steps of removing a portion of bone marrow containing hematopoietic stem cells from a subject prior to receiving anti-cancer therapy; maintaining the cells in culture; contacting the hematopoietic stem cells with an effective amount of a compound comprising JLP or biologically active fragment, derivative, homolog or analog thereof, such that the cells do not undergo apoptosis when subjected to apoptosis-inducing environmental stress from the anti-cancer treatment; reintroducing the cells into the subject; and optionally administering to the subject at least one further anti- cancer treatment.

The invention also provides methods of identifying a compound which alters the association of JLP with components of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module, comprising the steps of providing a compound comprising JLP or biologically active fragment, derivative, homolog or analog thereof, contacting the JLP compound with a signaling pathway component and a test compound, and determining whether the test compound alters association of the signaling pathway component with the JLP compound.

Amino Acid Abbreviations

The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, is understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule:

A Alanine Ala

C Cysteine Cys

D Aspartic Acid Asp

E Glutamic Acid Glu

F Phenylalanine Phe

G Glycine Gly

H Histidine His

I Isoleucine He

K Lysine Lys

L Leucine Leu

M Methionine Met

N Asparagine Asn

P Proline Pro

Q Glutamine Gin R Arginine Arg

S Serine Ser

T Threonine Thr

N Naline Nal w Tryptophan Trp

Y Tyrosine Tyr

Definitions A "heterologous" nucleic acid or peptide sequence is a sequence not naturally associated with a host cell into which it is introduced, including non- naturally occurring multiple copies of a naturally occurring sequence. When referring to two sequences, the term means that the two sequences have different origins; for example, a mouse nucleic acid coding sequence and a nucleic acid sequence encoding an HIN TAT protein transduction domain are heterologous sequences with respect to each other.

A "nucleic acid molecule" or "nucleic acid sequence" is a segment of single- or double-stranded DΝA or RΝA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DΝA.

"Expression vector" as used herein means a nucleic acid sequence, for example a plasmid, capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleic acid sequence. An expression vector comprising the nucleic acid sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression vector may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression vector is heterologous with respect to the host; i.e., the particular nucleic acid sequence of the expression vector does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transfection event. The expression of the nucleic acid sequence in the expression vector may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus (e.g., radiation). In the case of a multicellular organism, the promoter can also be specific to a particular tissue, organ or stage of development.

"Operably linked" refers to two or more nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be "operably linked" to a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.

A "coding sequence" is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, hnRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism or cell to produce a protein.

A "promoter" is an untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression. "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. Regulatory elements also typically encompass sequences required for proper transcription of the nucleotide sequence. "Transfection" is a process for introducing isolated nucleic acid into a host cell or organism. The nucleic acid molecule can be stably integrated into the genome of the host cell or organism, or the nucleic acid molecule can be present as an extrachromosomal molecule. Transfected cells or organisms are understood to encompass not only the end product of a transfection process, but also progeny thereof containing the transfected nucleic acid.

"Transgenic" or "recombinant" refer to a host organism or cell into which a heterologous nucleic acid molecule has been transfected. A "non- transfected" "non-transgenic," or "non-recombinant" host refers to a wild-type orgamsm or cell which does not contain the transfected nucleic acid molecule.

"Antibody" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered or removed from the natural state through the actions of a human being. For example, a nucleic acid sequence or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid sequence or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

The expression "amino acid" as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. "Standard amino acid" means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, "synthetic amino acid" also encompasses chemically modified amino acids, including salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide' s circulating half life without adversely affecting their biological activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention.

Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.

As used herein, "protecting group" with respect to a terminal amino group of a peptide means any of the various amino-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, acyl protecting groups such as formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups such as benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert- butoxycarbonyl or adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting groups.

As used herein, "protecting group" with respect to a terminal carboxy group of a peptide means any of various carboxyl-terminal protecting groups traditionally employed in peptide synthesis. Such protecting groups include, for example, tert-butyl, benzyl or other acceptable groups linked to the terminal carboxyl group through an ester or ether bond.

"Carboxy-terminal truncation fragment" with respect to an amino acid sequence means a fragment obtained from a parent sequence by removing one or more amino acids from the carboxy-terminus thereof.

"Derivative" includes any purposefully generated peptide which in its entirety, or in part, has a substantially similar amino acid sequence to JLP. Derivatives of JLP may be characterized by single or multiple amino acid substitutions, deletions, additions, or replacements. These derivatives include (a) derivatives in which one or more amino acid residues of JLP are substituted with conservative or non-conservative amino acids; (b) derivatives in which one or more amino acids are added to JLP; (c) derivatives in which one or more of the amino acids of JLP includes a substituent group; (d) derivatives in which JLP or a portion thereof is fused to another peptide (e.g., serum albumin or protein transduction domain); (e) derivatives in which one or more nonstandard amino acid residues (i.e., those other than the 20 standard L-amino acids found in naturally occurring proteins) are incorporated or substituted into the JLP sequence; and (f) derivatives in which one or more nonamino acid linking groups are incorporated into or replace a portion of JLP.

A "homolog" of JLP includes any nonpurposely generated peptide which in its entirety, or in part, has a substantially similar amino acid sequence to JLP. Homologs may include paralogs, orthologs, and naturally occurring alleles or variants of JLP .

An "analog" of JLP includes any non-peptide molecule comprising a structure that mimics the physico-chemical and spatial characteristics of JLP, and is biologically active.

"Biologically active," with respect to JLP, or fragments, derivatives, homologs and analogs of JLP means the ability of the compound to associate with one or more components of the MEKK3-MKK4-JNK/p38 MAPK/c- Myc/MAX signaling module, or exhibiting immunogenic characteristics of a JLP epitope.

A compound "associates" with one or more components of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module when the compound co-precipitates with one or more of the signaling module components under the conditions outlined in Noguchi et al. (1999), J. Biol. Chem. 274: 32580-32587 and Example 2 below (for association in vivo), or as outlined in Blanar and Rutter (1992), Science 256: 1024-1018 and Example 3 below (for association in vitro).

For example, in vivo association of the present compounds with MEKK3-MKK4-JNK p38 MAPK/c-Myc/MAX signaling module components can be shown by expressing the compound of interest as an S-tagged compound in COS7 cells. One or more signaling module components are also expressed in the COS7 cells; these components are tagged with either hemagglutinin (HA), FLAG or another appropriate tag. Two days after transfection with vectors expressing the S-tagged compound and tagged signaling module component(s), COS7 cells expressing the tagged proteins are incubated with cleavable cross- linking reagent dithiobis(succinimidylpropionate) (DSP; Pierce) at 1.5 mg/ml at 37 °C for 30 minutes, as described in Noguchi et al., supra. Cells are washed with PBS and lysed in 400μl buffer S [250mM Tris-HCl (pH 7.5); 137mM NaCl; 1% NONIDET P-40; 0.1% SDS; 0.5% sodium deoxycholate; ImM phenylmethylsulfonyl fluoride; 2μg/ml pepstatin; 2μg ml leupeptin; 1.9μg/ml aprotinin; ImM Na₃NO ], followed by brief sonication. The sonicated cell lysates are kept on ice for 20 minutes and centrifuged to clear cell debris. The protein concentrations of the lysates are determined, and samples from each cell lysate containing 400 to 500μg protein are mixed with the 50μl S-protein agarose bead slurry (Νovagen) at 4 °C for 1 hr in a rotator at 12 rpm. Each sample is then washed four times with Buffer S and centrifuged at 16,000g for 15 seconds. The bead/protein precipitates are incubated with 50μl IX sample buffer (2% SDS; 0.5% β-mercaptoethanol; 60mM Tris-HCl pH 6.8; 10% glycerol; 0.001% Bromophenol Blue) at 37 °C for 30 min., then boiled for 7 min. The boiled samples are electrophoresed in a 10% SDS-PAGE gel for 2 hours at 180 volts, followed by transfer onto nitrocellulose filters for Western blot analysis. Western blot analysis is performed by blocking the transferred filters with 5% low-fat milk in TBST (20mM Tris-HCl (pH 7.5); 200mM ΝaCl; 0.1% Tween-20) and hybridizing with antibodies specific to the tag on the signaling module component (e.g. anti-HA or anti-FLAG). After hybridization, the filters are washed with TBST and incubated with secondary antibodies conjugated with horseradish peroxidase in 5% low-fat milk/TBST. The filters are then washed with TBST, and specific signals detected with the Renaissance Western blot chemiluminescence reagent (ΝEΝ).

Determination of in vitro association of compounds with components of the MEKK3-MKK4-JΝK/p38 MAPK/c-Myc/MAX signaling module can be performed as follows: 5 nM of a test compound and 5 nM of at least one signaling module component are mixed and incubated at 4 °C for 30 min. to allow formation of a test compound/signaling component complex. The complex is immunoprecipitated with pre-immune serum or an antibody specific to the signaling component in 250 microliters total volume of HEGKD buffer (20 mM HEPES-KOH, pH 7.7; O.lmM EDTA; 2.5mM MgCl₂; 75mM KC1; 0.05% NONIDET P-40 and ImM dithiothreitol) at 4 °C for 1 hour. Protein A Sepharose (for rabbit antibodies) or Protein G Sepharose (for mouse antibodies) at a 1:1 v/v slurry is added, and the mixture is incubated at 4 °C for another hour. The immunocomplexes were washed four times with HEGKD buffer and resolved by SDS-PAGE, followed by transfer of the complexes onto nitrocellulose membranes for Western blot analysis with the component-specific antibody. Alternatively, either the compound or the signaling pathway component can be radiolabeled prior to immunoprecipitation, for example by radioiodination with ¹²⁵I, and the immunocomplexes detected by autoradiography.

A compound which "exhibits immunogenic characteristics of a JLP epitope" means that the compound 1) elicits a specific humoral or cellular immune response in a mammal to an epitope of JLP. As used herein, an "epitope" is a distinct structural area of an immunogen that can combine with an antibody or T-lymphocyte receptor. Reactivity to JLP epitopes may be determined by known immunological techniques, such as immunoprecipitations and Western blot analyses as described above and in the Examples. By way of illustration, a compound exhibiting immunogenic characteristics of a JLP epitope will, on injection into a mouse, cause that mouse to develop antibodies that react with JLP as detected, for example, by Western blot or enzyme-linked immunosorbent assay.

By "libraries" is meant pools and subpools of compounds, for example fragments, derivatives, homologs, analogs, or pro-analogs of JLP. "Peptide" and "protein" are used interchangeably, and refer to a compound comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds (e.g., peptide isosteres). No limitation is placed on the maximum number of amino acids which may comprise a protein or peptide. The amino acids comprising the peptides or proteins described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred. The amino acid comprising the peptides or proteins described herein may also be modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide. Also, a given peptide may contain many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, ga ma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylatiori, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62, the entire disclosures of which are herein incorporated by reference.

"Variant" as the term is used herein, is a nucleic acid sequence or peptide that differs from a reference nucleic acid sequence or peptide respectively, but retains essential properties. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or it can be a variant that is not known to occur naturally. Non- naturally occurring variants of nucleic acids and peptides can be made by mutagenesis techniques or by direct synthesis.

As used herein, a peptide or a portion of a peptide which has a "substantially similar amino acid sequence" to JLP means the peptide, or a portion thereof, has an amino acid sequence identity or similarity to JLP of greater than about 70%. Preferably, the sequence identity is greater than about 75%, more preferably greater than about 80%, particularly preferably greater than about 90%, and more particularly preferably greater than about 95%, and most preferably greater than about 98%. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm; BLASTP and TBLASTN settings to be used in such computations are indicated in Table 1 below. Amino acid sequence identity is reported under "Identities" by the BLASTP and TBLASTN programs. Amino acid sequence similarity is reported under "Positives" by the BLASTP and TBLASTN programs. Teclmiques for computing amino acid sequence similarity or identity are well known to those skilled in the art, and the use of the BLAST algorithm is described in Altschul et al. (1990), J. Mol. Biol. 215: 403-10 and Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402, the entire disclosures of which are herein incorporated by reference. BLASTP and TBLASTN programs utilizing the BLAST 2.0.14 algorithm are available, for example, at the National Center for Biotechnology Information World Wide Web site BLAST server. Table 1 - Settings to be used for the computation of amino acid sequence similarity or identity with BLASTP and TBLASTN programs utilizing the BLAST 2.0.14 algorithm.

*The SEG program is described by Wootton and Federhen (1993), Comp t. Chem. 17: 149-163.

"Substantially similar nucleic acid sequence" means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not affecting the peptide function occur. Preferably, the substantially similar nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is, for example, at least 70%, Preferably, the sequence identity is greater than about 75%, more preferably greater than about 80%, particularly preferably greater than about 90%, and more particularly preferably greater than about 95%, and most preferably greater than about 98%. Substantial similarity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50 °C; preferably in 7% (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 1XSSC, 0.1% SDS at 50 °C, more preferably 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 0.5.XSSC, 0.1% SDS at 50 °C; and most preferably in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 0.1XSSC, 0.1% SDS at 65 °C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, but are not limited to: GCS program package (Devereux et al. (1984), Nucl. Acids Res. L2: 387), and the BLASTN or FASTA programs (Altschul et al. (1990), J. Mol. Biol. 2 5: 403). The default settings provided with these programs are adequate for determining substantial similarity of nucleic acid sequences for purposes of the present invention. "Substantially purified" refers to a peptide or nucleic acid sequence which is substantially homogenous in character due to the removal of other compounds (e.g., other peptides, nucleic acids, carbohydrates, lipids) or other cells originally present. "Substantially purified" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or formulation into a pharmaceutically acceptable preparation.

"Synthetic mutant" includes any purposefully generated mutant or variant derived from JLP. Such mutants may be purposefully generated by, for example, chemical mutagenesis, polymerase chain reaction (PCR) based approaches, or primer-based mutagenesis strategies well known to those skilled in the art.

Brief Description of the Figures

FIG. IA shows the deduced amino acid sequence of mouse JLP.

Conserved domains A, B and C are boxed. The heptad repeat of leucine and homologous hydrophobic amino acids in LZI and LZII are circled. The putative SH2 and SH3 binding sites are shaded and underlined, respectively. FIG. IB shows the deduced amino acid sequence of human JLP. Conserved domains A, B and C are boxed. The heptad repeat of leucine and homologous hydrophobic amino acids in LZI and LZII are circled. The putative SH2 and SH3 binding sites are shaded and underlined, respectively. FIG. 1C shows a Western blot analysis of JLP overexpressed in COS7 cells and endogenous JLP from 32Dcl3 cells.

FIG. 2A shows Western blots of S-protein agarose precipitates from lysed COS7 cells expressing HA-tagged JNK1 (HA- JNK); FLAG-tagged p38 MAPKα (Flag-p38) or ERK2 (ERK), with or without S-tagged JLP (JLP-S). The blots were probed with antibodies against the S-tag, the HA-tag, the FLAG- tag or ERK2. COS7 cells transfected with expression vector alone (V) were used as a control. FIG. 2B (upper panel) is a schematic of JLP C-terminal deletion mutants listed in Table 2, and (lower panel) a Western blot of S-protein agarose precipitates from lysed COS7 cells co-expressing HA-JNKl with expression vector alone (V), full-length JLP ("1307"), or JLP deletion mutants. All JLP were S-tagged. The blot was probed with HA-specific antibody. FIG. 2C (right panel) is a schematic of JLP N-terminal fragments listed Table 3, and (left panel) a Western blot analysis of S-protein agarose precipitates from lysed COS7 cells co-expressing HA-JNKl with expression vector alone (V) or the N- terminal JLP fragments listed in Table 3. All JLP were S-tagged. The blot was probed with HA-specific antibody. The levels of JLP mutants on the Western blot as detected by the anti-S-tag antibody is also shown as a loading control. FIG. 2D shows a Western blot of S-protein agarose precipitates from lysed COS7 cells co-expressing FLAG-tagged p38 MAPKα with expression vector alone (V) or JLP fragments listed in Table 3. All JLP were S-tagged. The precipitates were analyzed with FLAG-specific antibody. FIG. 2E shows a Western blot of a COS7 cell lysate immunoprecipitated with JNK-1 specific antibody (JNK) or a control rabbit antibody (C). The blots were probed with JLP C-terminal specific antibody JLP.

FIG 3A shows a Western blot of recombinant Max protein mixed with recombinant M2 and immunoprecipitated with pre-immune serum (PI) or Max- specific antibody (Max Ab). The blot was probed with JLP N-terminal specific antibody. FIG. 3B (upper panel) is a schematic of recombinant Max mutants, and (middle panel) a Western blot of recombinant Max mutants mixed with recombinant M2 and immunoprecipitated with Max-specific antibody (Max Ab). The last lane labeled M2 contains recombinant M2 loaded directly onto the gel. The lower panel is a Coomassie blue stained gel of the wild-type and mutant Max proteins (2 μg each) used for the Western blot, shown as a loading control. FIG. 3C shows a Western blot of recombinant wild-type Max mixed with either recombinant M2 or recombinant M2LZI, and immunoprecipitated with pre-immune serum (PI) or Max-specific antibody (Max Ab). FIG. 3D shows a Western blot of S-protein agarose precipitates from lysed COS7 cells transiently transfected to express Max with or without S-tagged JLP (JLP-S). COS7 cells transfected with expression vector alone (V) were used as a control. The blot was probed with S-tag or Max specific antibodies. FIG. 3E show an electrophoretic mobility shift assay of recombinant c-Myc and Max mixed with P-labeled CM1 probe in the presence or absence of recombinant M2. The DNA/protein complexes were resolved in a native non-denaturing polyacrylamide gel (4%), dried and subjected to autoradiography.

FIG. 4A shows a Western blot of S-protein agarose precipitates from lysed COS7 cells transiently transfected to express c-Myc with or without S- tagged JLP (JLP-S). COS7 cells transfected with expression vector alone (V) were used as a control. The blot was probed with S-tag or c-Myc specific antibodies. FIG. 4B shows Western blots of S-protein agarose precipitates of lysed COS7 cells co-expressing c-Myc with the expression vector alone (V), or the N-terminal JLP fragments listed in Table 3. All JLP were S-tagged. The blots were probed with c-Myc specific antibody.

FIG. 5A shows Western blots of S-protein agarose precipitates from lysed COS7 cells transiently transfected to express GST-tagged MKK4 (GST- MKK4), HA-tagged MEKK3 (HA-MEKK3), and HA-tagged MKK3 (HA- MKK3) with or without S-tagged JLP (JLP-S). The blots were probed with S- tag, HA-tag or GST-tag specific antibodies. FIG. 5B shows a Western blot of S-protein agarose and glutathione-Sepharose precipitates from lysed COS7 cells transiently transfected to express HA-JNKl and GST or GST-tagged MKK4 (GST-MKK4), with or without JLP-S, as indicated. The blots were probed with HA-tag or GST-tag specific antibodies.

FIG. 6 A shows a Western blot of cell lysates from RatlA cells stably transfected with pOPI3 vector alone (V) or pOPI3 vector encoding JLP (JLP). The blot was probed with an N-terminal-specific JLP antibody. For pOPI3-JLP vector-transfected cells, JLP levels were determined in the presence or absence of IPTG. FIG. 6B shows a light micrograph of the cells from FIG. 6A were treated with UV radiation (254 nm, 5 min) to induce apoptosis, and the viability of the cells monitored by staining with trypan blue at 0, 12, 23 and 48 hr. FIG 6C is a plot of the number of viable cells (mean + SD) from three independent replicates of cells treated as in FIG. 6B.

FIG. 7 shows Western blots of S-protein agarose precipitates from lysed NIH-3T3 cells transiently transfected to express JNK1-S, FLAG-p38 MAPKα, HA-MKK4, and HA-MEKK3. The cells were also transfected with wild type JLP-HA (WT), JLP dominant negative mutant lacking the JNK binding domains (ΔJLP), or an empty expression vector (V), in the presence or absence of a dominant positive mutant of MEKK1 (ΔMEKK). The blots were probed with an antibody specific for phospho-JNK.

Detailed Description of the Invention

A scaffolding protein named JLP (for JNK-associated leucine zipper protein), and nucleic acid sequences encoding the protein have been isolated and substantially purified from both mouse and human. JLP tethers MEKK3, MKK4, JNK, p38 MAPK, c-Myc and MAX into a signaling module which controls the cellular apoptotic response. JLP therefore functions as a signaling conduit to transmit extracellular signals to the nucleus through MEKK3-MKK4- JNK/p38 MAPK/c-Myc/MAX signaling module. The isolated human and mouse JLP cDNA sequences are given in SEQ

ID NO: 1 and SEQ ID NO: 3, respectively. These cDNA's show a single large open reading frame ("ORF") of approximately 3.9 kilobases comprising 1307 codons in the mouse and 1311 codons in the human.

The invention is not limited to the nucleotide sequences of SEQ ID NOS: 1 and 3, but also includes fragments thereof. It is preferred that the fragments be uniquely found within the JLP cDNAs. As it is conventionally understood that the smallest nucleotide sequence which is unlikely to be found in more than one segment of the mammalian genome is at least about 18 nucleotides in length, certain preferred fragments of the invention are at least 18 contiguous nucleotides in length, more preferably at least 25 contiguous nucleotides in length. It is understood by one or ordinary skill in the art, however, that any unique fragment of contiguous nucleotides within the JLP cDNA sequences is within the scope of the invention. If, for example, it is found that a particular 18 contiguous nucleotide fragment is not unique, the length of the fragment can be incrementally increased one or more contiguous nucleotides at a time until the fragment is unique or sufficiently unique to achieve the desired ends. Thus, this disclosure expressly describes fragments having any length of contiguous nucleotides from 18 to 4675 of SEQ ID NO: 1 and fragments having any length of contiguous nucleotides from 18 to 4667 of SEQ ID NO: 3.

It is also understood by one of ordinary skill in the art that a plurality of different short fragment lengths may be employed in combination to achieve the same or similar effect as a single longer fragment. Thus, two fragments from different parts of the complete JLP cDNA sequences, each being as short as, e.g., 6 contiguous nucleotides each, can be used in combination as probes to specifically detect the presence of the complete sequences' complementary strand in a sample. Such fragments need not be individually unique.

It is particularly preferred that the nucleic acid fragments of the invention encode a peptide having JLP binding activity or immunologic properties of JLP, as described in more detail below.

In addition to the complete JLP cDNA sequences and fragments thereof, the invention also encompasses complementary sequences thereto, and homologous nucleic acid sequences substantially similar to the complete sequence, the fragments and/or the complements. These sequences include DNA, RNA and analogues thereof, including peptide nucleic acids.

Substantially similar nucleic acid sequences of the invention encode peptides that are the same or similar to JLP, or fragments, derivatives or homologs of JLP. One of ordinary skill can readily identify other nucleic acid sequences which encode JLP based on substantial similarity to SEQ ID NOS: 1 and 3. Nucleic acid sequences that exhibit substantial similarity to SEQ ID NOS: 1 and 3 can be considered JLP nucleic acid sequences according to the present invention.

The nucleotide sequences described herein can be used to produce recombinant JLP amino acid sequences useful in the methods of the invention.

The invention also provides novel, isolated JLP amino acid sequences.

JLP is a ubiquitously expressed cytoplasmic protein with a punctate distribution

(see Example 1 below). The ubiquitous expression pattern of JLP means it can function as a scaffolding protein for the ubiquitously expressed JNK and p38 MAP kinases, such as JNKl, JNK2, p38 MAPKα and p38 MAPKβ, in virtually all cell types.

The primary amino acid sequence predicted from the mouse JLP cDNA open reading frame is 1307 amino acids long (see SEQ ID NO: 4), and the encoded protein is expected to have a molecular weight of 143 kDa. However, mouse JLP overexpressed in COS7 cells showed an apparent molecular weight of 180 kDa by Western blot analysis. This 180 kDa protein is identical to endogenous mouse JLP immunoprecipitated from 32Dcl3 cells (see FIG. 1C). Without wishing to be bound by any theory, it is believed that JLP undergoes one or more forms of post-translational modification in vivo, which accounts for the increase in apparent molecular weight.

A sequence analysis of the mouse JLP is presented in FIG. IA. As can be seen from the figure, Mouse JLP contains two leucine zipper domains (LZI and LZII) and a C-terminal domain with extensive homology to Caenorhάbditis elegans protein ZK1098.10 (GenBank accession no. Z22176). The heptad repeat of the LZI and LZII leucine and homologous hydrophobic amino acids are circled in FIG. IA. Mouse JLP contains three putative SH2 and SH3 binding sites (shaded and underlined, respectively, in FIG. IA) which can potentially tether SH2- and SH3 -containing kinases to the JLP signaling module. JLP may therefore provide a link to other signaling pathways mediated by SH2/SH3 -containing kinases. Mouse JLP also has three domains which are conserved relative to C. elegans protein ZK1098.10, labeled as A, B and C and boxed in FIG. IA. A similar sequence analysis of the human JLP is presented in , FIG. IB. Human JLP contains two leucine zipper domains (LZI and LZII); the heptad repeat of the LZI and LZII leucine and homologous hydrophobic amino acids are circled. The C-terminal domain has extensive homology to C. elegans protein ZK1098.10. Human JLP contains three putative SH2 and two SH3 binding sites (shaded and underlined, respectively, in FIG. IB). Human JLP also has three domains which are conserved relative to C. elegans protein ZK1098.10, labeled as A, B and C and boxed in FIG. IB.

JLP shares approximately 69% amino acid identity with JSAP1 and JIP3, which are two known mammalian scaffolding proteins for the JNK- signaling pathway. However, JASP1 and JIP3 do not appear to contain a leucine-zipper corresponding to LZI of JLP. Thus, JASP1 and JIP3 are likely unable to form heterodimers with Max or other transcriptional factors via that domain. Moreover, unlike JLP, JSAPl and JIP3 cannot associate with both JNK and p38 MAP kinases.

The association of JLP with the various components of the MEKK3- MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module is discussed below with respect to the mouse protein. However, it is understood that human JLP has substantially the same characteristics and biological activities. It is understood that, unless otherwise indicated, that reference to a biological molecule by name in the following discussion (e.g., "JLP", "JNKl", etc.) means the expressed protein and not the nucleic acid encoding the protein. JNK/&38 MAP Kinase association JLP specifically associates with JNK via the N-terminal region at two binding sites comprising JLP amino acids 1 - 110 and 160 - 209, which are located at the N-terminus and between leucine-zipper domains LZI and LZII, respectively. These binding sites are located between the two leucine-zipper domains LZI and LZII. See FIG. 2A - 2C and Example 2 below. JNK binding to JLP appears to occur at either of these binding sites. JLP also specifically associates with p38 MAP kinase at amino acids 1 - 110 or 160 - 209, and again either site may bind p38 MAP kinase. See FIG 2D and Example 2 below. JLP thus has dual binding sites (at amino acids 1 - 110 and 160 - 209) for

JNK and p38 MAPK. This arrangement likely allows JNK or p38 MAP kinase to phosphorylate factors binding to the nearby LZI and LZII (or other) regions on JLP. Moreover, a single JLP molecule can simultaneously tether both JNK and p38 MAPK, allowing the simultaneous activation of both MAP kinases.

JLP does not interact with ERK2, as evidenced by a lack of JLP/ERK2 co-precipitation under conditions in which the interaction between JLP and JNKl or p38 MAPKα was readily seen. JLP is thus distinct from JSAP1 and JIP3, which are known to associate with JNK but not with p38 MAPKα. c-Myc/MAX association c-Myc associates with JLP in the N-terminal region at two binding sites comprising amino acids 1 - 110 and 160 - 209, which are located at the N- terminus and between leucine-zipper domains LZI and LZII, respectively (see FIG. 4B, and Example 3 below). However, neither LZI or LZII appear to play a role in the association of JLP with c-Myc.

As seen in FIGS. 2C and 4B, c-Myc and JNKl bind to the same regions of JLP. Without wishing to be bound by any theory, this suggests that either JNKl and c-Myc bind to the same domain or the Myc association with JLP is mediated by JNK. JLP binds to Max via the first leucine zipper motif (LZI) in the N- terminal end of the protein. See FIGS. 3 A and 3C, and Example 3 below. Immunoprecipitation experiments carried out with JLP fragment containing either an altered LZI or altered LZII motif confirmed that LZI is important for the interaction with Max (See FIG. 3C). The second leucine-zipper motif LZII does not appear to participate in Max binding.

Electrophoretic mobility shift assays using the putative Max target nucleotide sequence CACGTG (the "E-box") indicate that JLP has a high affinity for Max/ Max homodimers, and that JLP interaction with Max results in the dissociation of these homodimers. However, the affinity of JLP for Max appears to be weaker than that of c-Myc, as evidenced by the inability of JLP to dissociate c-Myc/Max heterodimers. See FIG. 3E and Example 3 below. The cytoplasmic localization of JLP and its ability to interact with transcription factors like c-Myc and Max indicates that JLP may associate with these proteins as they are nascently translated in cytoplasm. JLP may also disrupt endogenous Max homodimers to form JLP/Max heterodimers. Moreover, c-Myc and Max are put in close proximity on JLP where the higher affinity of c-Myc for Max should favor dissociation of the Max/JLP heterodimers and the formation of c-Myc/Max heterodimers. Without wishing to be bound by any theory, this may be a mechanism by which c-Myc/Max heterodimers are formed following their activation in vivo. MEKK3/MKK4 association

The binding sites of MEKK3 and MKK4 on JLP have not been identified, but specific interaction of JLP with these proteins has been shown via immunoprecipitation assays (see FIG. 5 and Ex. 4 below). JLP also binds simultaneously to both JNK and MKK4 (which is an upstream kinase of JNK), as shown in FIG. 5B and Ex. 4 below.

Activation of JNK by JLP

JLP activates JNK in the presence of p38MAPK, MKK4 and MEKK3.

See, e.g., FIG. 7 and Example 6 below, which shows that a dominant negative mutant of JLP lacking the JNK binding sites (ΔJLP) abolished JNK phosphorylation under conditions which typically result in constitutive activation of the JNK and p38MAPK pathways

JLP, or biologically active fragments, homologs and derivatives thereof, can comprise natural or synthetic peptides produced by any known means, including synthesis by biological systems and by chemical methods.

Biological synthesis of peptides is well known in the art, and includes the transcription and translation of a synthetic gene encoding JLP or biologically active fragments, homologs, and derivatives thereof. Chemical peptide synthesis includes manual and automated techniques well known to those skilled in the art.

For example, the JLP coding sequences of SEQ ID NOS: 1 and 3 can be subcloned into an appropriate plasmid expression vector for propagation and expression in an appropriate host. The techniques used to isolate or construct nucleic acid sequences, construct plasmid expression vectors, transfect host cells, and express a nucleic acid sequence of interest are widely practiced in the art, and practitioners or ordinary skill are familiar with the standard resource materials which describe specific conditions and procedures. For example, general methods for the cloning and expression of recombinant molecules are described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratories, 1982; and in Ausubel, Current Protocols in Molecular Biology, Wiley and sons, 1987, the disclosures of which are incorporated herein by reference.

JLP produced from an expression vector may be obtained from the host cell by cell lysis, or by using heterologous signal sequences fused to the protein which cause secretion of the protein into the surrounding medium. Preferably, the signal sequence is designed so that it may be removed by chemical or enzymatic cleavage, as is known in the art. The JLP thus produced may then be purified in a manner similar to that utilized for isolation of JLP from natural sources.

The JLP peptides and fragments of the present invention may be synthesized de novo using conventional solid phase synthesis methods. In such methods, the peptide chain is prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group; various coupling reagents e.g., dicyclohexylcarbodiimide or carbonyldimidazole; various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide; and the various cleavage reagents, e.g., trifluoroactetic acid (TFA), HCI in dioxane, boron tris-(trifluoracetate) and cyanogen bromide; and reaction in solution with isolation and purification of intermediates are methods well-known to those of ordinary skill in the art. A preferred peptide synthesis method follows conventional Merrifield solid phase procedures well known to those skilled in the art. Additional information about solid phase synthesis procedures can be had by reference to Steward and Young, Solid Phase Peptide Synthesis, W.H. Freeman & Co., San Francisco, 1969; the review chapter by Merrifield in Advances in Enzymology 32:221-296, (Nold FF, ed.), Interscience Publishers, New York, 1969; and Erickson and Merrifield (1990), The Proteins 2:61-64, the entire disclosures of which are incorporated herein by reference. Crude peptide preparations resulting from solid phase syntheses can be purified by methods well known in the art, such as preparative HPLC. The amino-terminus can be protected according to the methods described for example by Yang et al, FEBS Lett. 272:61-64 (1990), the entire disclosure of which is herein incorporated by reference.

Automated peptide synthesis to produce proteins comprising SEQ ID NOS: 2 and 4 can be performed with commercially available peptide synthesizers. Biologically active fragments according to the invention can also be obtained by the chemical or enzymatic fragmentation of larger natural or synthetic JLP peptides. Techniques to synthesize or otherwise obtain peptides and peptide fragments are well known in the art.

The JLP peptides and fragments can also comprise a label a (e.g., substances which are magnetic resonance active; radiodense; fluorescent; radioactive; detectable by ultrasound; detectable by visible, infrared or ultraviolet light) so that the compound may be detected. Suitable labels include, for example, fluorescein isothiocyanate (FITC); peptide chromophores such as phycoerythrin or phycocyanin and the like; bioluminescent peptides such as the luciferases originating from Photinus pyrali; fluorescent proteins originating from Renilla reniformi; and radionuclides such as P, P, S, I or I. For example, the label can comprise an NH₂-terminal fluorescein isothiocyanate (FITC)-Gly-Gly-Gly-Gly motif that is conjugated to a protein transduction domain.

Methods of modifying peptide sequences with labels are well known to those skilled in the art. For example, methods of conjugating fluorescent compounds such as fluorescein isothiocyanate to short peptides are described in Danen et al., Exp. Cell Res., 238:188-86 (1998), the entire disclosure of which is incorporated herein by reference. The present invention also provides biologically active derivatives of JLP. The techniques for obtaining these derivatives are known to persons having ordinary skill in the art and include, for example, standard recombinant nucleic acid techniques, solid phase peptide synthesis techniques and chemical synthetic techniques as described above. Linking groups may also be used to join or replace portions of JLP and other peptides. Linking groups include, for example, cyclic compounds capable of connecting an amino-terminal portion and a carboxyl terminal portion of JLP. Techniques for generating derivatives are also described in U.S. patent 6,030,942 the entire disclosure of which is herein incorporated by reference (derivatives are designated "peptoids" in the 6,030,942 patent). JLP derivatives can also incorporate labels such as are described above into their structure.

Examples of derivatives according to the present invention include, for example, synthetic variants of JLP. JLP Derivatives also include, for example, fusion peptides in which a portion of the fusion peptide has a substantially similar amino acid sequence to JLP. Such fusion peptides can be generated by techniques well-known in the art, for example by subcloning nucleic acid sequences encoding JLP and a heterologous peptide sequence into the same expression vector, such that the JLP and heterologous sequences are expressed together in the same protein. For example, the heterologous sequences can comprise a peptide leader sequence that directs entry of the protein into a cell. Such leader sequences include "protein transduction domains" or "PTDs," as discussed in more detail below.

The present invention also provides biologically active homologs of JLP. Homologs have substantially similar amino acid sequence and binding activity to JLP and can be identified on this basis. JLP homologs can also incorporate labels such as are described above into their structure.

The present invention also provides biologically active analogs of JLP. Such analogs can, for example, be small organic molecules capable of binding one or more components of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module. JLP analogs can incorporate labels such as are described above into their structure. JLP analogs preferably comprise a structure, called a pharmacophore, that mimics the physico-chemical and spatial characteristics of JLP. Consequently, pro-analogs can, for example, be designed based on variations in the molecular structure of JLP binding sites or portions of JLP. The structure and function of various portions of JLP can be determined, for example, through the co-immunoprecipitation analyses set forth in the Examples. Alternatively, JLP structure and function can be determined using nuclear magnetic resonance (NMR), crystallographic, or computational methods which permit the electron density, electrostatic charges or molecular structure of certain portions of JLP or fragments thereof to be mapped.

The solid-phase synthesis methods described by Schreiber (2000), Science 287:1964-1969, the entire disclosure of which is herein incorporated by reference, can be used to generate a library of distinct pro-analogs generated by organic syntheses. Briefly, a suitable synthesis support, for example a resin, is coupled to a pro-analog precursor and the pro-analog precursor is subsequently modified by organic reactions such as, for example, Diels-Alder cyclization. The immobilized pro-analog is then be released from the solid substrate. Pools and subpools of pro-analogs can be generated by automated synthesis techniques in parallel, such that all synthesis and resynthesis can be performed in a matter of days. Such pools and subpools of pro-analogs are said to comprise libraries. Once generated, pro-analog libraries can be screened for analogs; i.e. compounds exhibiting the ability to bind to one or more components of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module, for example as described below in the Examples. Pro-analogs of JLP can also be designed, for example, by using the retrosynthetic, target oriented, or diversity-oriented synthesis strategies described by Schreiber (2000), supra. Retrosynthetic strategies, for example, require that key structural elements in a molecule be identified and then incorporated into the structure of otherwise distinct pro-analogs generated by organic syntheses. Here, key JLP structural elements have been identified; e.g., the leucine zipper domains LZI and LZII; conserved regions A, B and C; the SH2 and SH3 binding sites; and the binding sites for the signal module components. See FIG. 1 A and Examples 2 - 5 below.

U.S. patent 6,030,942, in particular Example 4 therein, describes retrosynthetic methods for the design and selection of analogs based on identified key structural elements in a protein, and is incorporated herein in its entirety.

The present invention also provides antibodies against JLP, or derivatives, homologs, analogs, or antigenic fragments thereof. The antibodies of the invention can, for example, specifically bind an epitope of JLP. The antibody can be a monoclonal antibody or a polyclonal antibody or an antibody fragment that is capable of binding antigen.

The antibodies of the invention can, for example, comprise antibodies, and preparations thereof, produced by immunizing an animal with substantially pure JLP or an immunogenic fragment thereof. The present invention includes chimeric, single chain, and humamzed antibodies, as well as Fab fragments and the products of a Fab expression library. Antibody fragments, such as Fab antibody fragments, which retain some ability to selectively bind to the antigen of the antibody from which they are derived, can be made using well known methods in the art. Such methods are generally described in U.S. patent 5,876,997, the entire disclosure of which is herein incorporated by reference.

Polyclonal antibodies can be generated against the compounds of the invention. Antibodies can be obtained following the administration of one or more peptides, fragments, derivatives, or homologs to an animal, using the techniques and procedures known in the art. Monoclonal antibodies can be prepared using the method of Mishell,

B.B. et al., Selected Methods In Cellular Immunology, (Freeman WH, ed.) San Francisco, 1980, the entire disclosure of which is herein incorporated by reference. Briefly, a peptide or peptide fragment of the present invention is used to immunize spleen cells of Balb/C mice. The immunized spleen cells are fused with myeloma cells. Fused cells containing spleen and myeloma cell characteristics are isolated by growth in HAT medium, a medium which kills both parental cells but allows the fused products to survive and grow. Thus in one embodiment, the invention comprises a hybridoma that produces a monoclonal antibody which specifically binds to a peptide or peptide fragment according to the invention.

Antibodies can be used to purify the compounds of the invention, using immunoaffinity techniques which are well known by those of skill in the art.

The compounds of the invention are useful in modulating the apoptotic response in cells under certain conditions. As used herein, "apoptotic response" refers to induction of genetically controlled cell death that is distinct from necrosis. Modulation of this response includes blocking or reversing apoptosis (i.e., inducing cell survival) as well as mitigating apoptosis (i.e., extending or delaying the time for apoptosis to occur).

Thus, the invention provides methods of regulating the apoptotic response in cells, comprising the steps of contacting the cells with an effective amount of compound comprising a JLP or biologically active derivative, homolog or analog thereof, such that the compound is introduced into the cell and said modulation is effected. In a preferred embodiment, the modulation of the apoptotic response comprises inducing cell survival.

As used herein, "contacting" a cell with a compound of the invention includes any method for introducing the present compounds into the cytoplasm of a cell, including transfection of nucleic acids encoding the present compounds into the cell such that the compounds are expressed therein, or directly introducing exogenous proteins or other compounds into the cell. Both cultured cells and cells in a living organism can be contacted with the present compounds. With regard to expression of the present compounds in a cell via a transfected nucleic acid, an "effective amount" means any amount which produces detectable expression of the compound within the cell. Expression of the present compounds can be detected by any known technique, such as PCR amplification of mRNA, Northern blot, or Western blot. Preferably, the cells are transfected with a nucleic acid sequence encoding a JLP, or biologically active derivative, homolog or analog of JLP, which comprises a plasmid expression vector. Such plasmids can be generated by recombinant nucleic acid and molecular cloning techniques well-known in the art, as discussed above.

Transfection methods for eukaryotic cells are well known in the art, and include, for example, direct injection of the nucleic acid into the nucleus or pronucleus; electroporation; liposome transfer; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; and transfection mediated by viral vectors. In a preferred method, the transfection is performed with a liposomal transfer compound, e.g., DOTAP (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate, Boehringer - Mannheim) or an equivalent, such as LIPOFECTIN. The amount of nucleic acid used is not critical to the practice of the invention; acceptable results may be achieved with 10 mM nucleic acid/10⁵ cells. A ratio of about 500 nanograms of plasmid vector in 3 micrograms of DOTAP per 10⁵ cells can be used.

Methods for the construction and propagation of plasmid vectors capable of expressing the present compounds, and techniques for transfecting such vectors into eukaryotic cells so that the compounds are expressed, are known in the art and are also described in the Examples below.

Contacting cells with a compound of the present invention can also comprise the direct introduction of the compounds into cells by methods known in the art. For cultured cells, such methods include administering the compounds either directly to the cells, or in the culture media. Preferably, an effective amount of a compound of the invention is included in fresh growth media which is periodically given to cells growing in culture. For example, a concentrated solution of the present compounds in a carrier such as sterile water, saline, growth media or the like can be diluted into fresh growth media prior to applying the fresh growth media to the cultured cells.

Cells in a living organism can be contacted with an effective amount of the present compounds by any parenteral or enteral means, such as direct injection into the tissue, injection into the vasculature, oral administration, and the like. A preferred method is the direct injection of the compound into the tissue. An "effective amount" of a compound comprising JLP, or a biologically active derivative, homolog or analog thereof which is directly introduced into a cell means any amount of the compound sufficient to cause the desired modulation in the apoptotic response. For example, in cultured cells, an effective amount can be between about 25 nM and about 300 nM, preferably at least 50 nM, more preferably at least 100 nM, and particularly preferably at least 150 nM. For in vivo administration, an effective amount can be, for example, between about 5g compound/kg and 25 g compound/kg, preferably at least lOg compound/kg, more preferably at least 20g compound/kg. In one embodiment, the present compounds are modified to enhance the uptake of the compounds into the cell. For example, the compounds can be encapsulated in a liposome prior to being contacted with the cells. The encapsulated compounds are delivered directly into the cells by fusion of the liposome to the cell membrane. Reagents and techniques for encapsulating the present compounds in liposomes are well-known in the art, and include, for example, the ProNectin™ Protein Delivery Reagent from Imgenex.

In another embodiment, the present compounds are modified to enhance their entry into a cell by associating the compounds with a peptide leader sequence known as a "protein transduction domain" or "PTD." These sequences direct entry of the compound into the cell by a process known as "protein transduction." See Schwarze et al. (1999), Science 285: 1569 - 1572, the entire disclosure of which is herein incorporated by reference. Proteins ranging in size from 15 to 120 kD have been transduced into a wide variety of human and murine cell types in vivo and in vitro using the PTD method. See Schwarze et al., supra; Nagahara et al. (1998), Nature Med. 4: 1449; Ezhevsky et al. (1997), Proc. Natl. Acad. Sci. U.S.A. 94: 10699; Lissy et al. (1998), Immunity 8: 57; and Gius et al. (1999), Cancer Res. 59: 2577, the entire disclosures of which are herein incorporated by reference in their entirety. Thus, in a further embodiment, the present compounds are introduced into the cytoplasm of cells by virtue of a PTD associated with the compound.

Without wishing to be bound by a particular theory, entry of exogenously added, PTD-associated compounds into the cell during protein transduction appears to occur in a rapid, concentration-dependent fashion. Moreover, the process appears to be receptor and transporter independent, see Derossi et al. (1996), J. Biol. Chem. 221: 18188, and may directly involve the lipid bilayer component of the cell membrane. Thus, all cell types, in particular mammalian cell types, are susceptible to protein transduction. Exogenous administration of PTD-linked compounds to cultured cells results in the rapid delivery of a roughly equal amount of the compound to each cell.

The present compounds can therefore be modified with a PTD that directs entry of the compound into the cell when the compound is administered exogenously to cells in culture or in vivo. A PTD can be located anywhere on the compound that does not disrupt the compound's biological activity. For compounds of the invention comprising a peptide, the PTD is preferably located at the N-terminal end.

PTDs are well-known in the art, and can comprise any of the known PTD sequences including, for example, arginine-rich sequences such as a peptide of nine to eleven arginine residues optionally in combination with one to two ly sines or glutamines as described in Guis et al. (1999), Cancer Res. 59: 2577-2580, the entire disclosure of which is herein incorporated by reference. Preferred are sequences of eleven arginine residues (SEQ ID NO: 5) or the NH₂- terminal 11 -amino acid protein transduction domain from the human immunodeficiency virus TAT protein (SEQ ID NO: 6). Other suitable leader sequences include other arginine-rich sequences; e.g., 9 to 10 arginines, or six or more arginines in combination with one or more lysines or glutamines. Such leader sequences are known in the art; see, e.g., Guis et al. (1999), supra. Preferably, the PTD is designed so that it is cleaved from the compound within the cell.

Kits and methods for constructing fusion proteins comprising a protein of interest (e.g., a JLP) and a PTD are known in the art; for example the TransNector™ system (Q-BIOgene), which employs a 16 amino acid peptide called "Penetratin™" corresponding to the Drosophila antennapedia DΝA- binding domain; and the Voyager system (Invitrogen Life Technologies), which uses the 38 kDa VP22 protein from Herpes Simplex Virus- 1. The compounds of the invention promote cell survival by bringing together the components of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module in a cell that either has been exposed to, or is in danger of being exposed to, an apoptosis-causing environmental stress. An apoptosis- causing environmental stress can be, for example, any stress which causes extensive DNA damage, such as ionizing radiation (e.g., UV or gamma radiation) or chemotherapeutics (e.g., anti-cancer drugs taxol, vincristine, vinblastine, and the like). Example 6 below shows that expression of JLP in mammalian cells provides protection from UV-induced apoptotic cell death. Thus, the invention provides a method of modulating the apoptotic response in cells by contacting a cell exposed to, or in danger of being exposed to, an apoptosis-inducing environmental stress with an effective amount of the compounds of the present invention, such that the cells do not undergo apoptosis in response to the environmental stress. Preferably, the cells do not undergo the stress-induced apoptosis for at least 12 hours, more preferably at least 24 hours, particularly preferably at least 48 hours, after exposure to the environmental stress.

In a preferred embodiment, cells are protected from undergoing apoptosis upon exposure to ionizing radiation, e.g. ultraviolet (UN) or gamma radiation, by contacting the cells with an effective amount of the present compounds.

For example, the compounds of the invention are useful in protecting normal bone marrow cells from radiologic or chemotherapeutic treatments designed to destroy primary or metastatic tumor cells in the body of a subject. In a typical course of cancer treatment, bone marrow containing hematopoietic stem cells is removed from the subject prior to administering ionizing radiation or an anti-cancer drug to the subject's body. After treatment, the bone marrow is re-introduced into the subject to replace those hematopoietic stem cells killed by the radiation or chemotherapy. However, the subject often needs additional radiation or chemotherapy treatments, and it is impractical to repeatedly remove, bank and re-introduce bone marrow cells each time the subject receives anti-cancer therapy. Thus, the present invention provides a method of protecting hematopoietic stem cells from apoptotic death induced by anti-cancer radiation or chemotherapy treatments, so that the hematopoietic stem cells can be left in the subject's body during treatment. This method comprises the steps of removing a portion of bone marrow containing hematopoietic stem cells from a subject prior to receiving anti-cancer therapy; maintaining the cells in culture and contacting the hematopoietic stem cells with an effective amount of a compound according to the present invention, such that the cells do not undergo apoptosis when subjected to apoptosis-inducing environmental stress; reintroducing the cells into the subject; and optionally administering to the subject at least one further anti-cancer treatment. '

Preferably, the hematopoietic stem cells are transfected with a nucleic acid encoding a compound of the invention, e.g. a plasmid expression vector, that stably integrates into the hematopoietic stem cell genome to provide long- term expression of the compound. Stable integration and expression can be confirmed by techniques known in the art, such as a Southern blot of hematopoietic stem cell genomic DNA using JLP cDNA or fragments thereof as a probe, or detection of JLP expression by Northern or Western blot. Once stable integration and/or expression of the JLP construct has been confirmed, the hematopoietic stem cells can be re-implanted in the subject, where they will continue to express the compound.

Alternatively, the nucleic acid encoding the compound can be placed under the control of an inducible promoter, such as a radiation-inducible promoter, so that JLP expression is initiated in the stem cells upon treatment with the anti-cancer agent. Radiation-inducible promoters are well known the art; see, for example, US Pat. Nos. 5,962,424 and 6,641,755, the entire disclosures of which are herein incorporated by reference. Suitable radiation inducible promoters for mammalian expression systems include the c-fos promoter; the c-jun promoter; the tumor necrosis factor alpha promoter; and vascular endothelial cell specific promoters such as the early growth response- 1 (Egr-1) promoter, in particular the CArG domain of the Egr-1 promoter; the ICAM-1 promoter; and the E-selectin gene promoter. Methods for isolating, identifying, separating, and culturing hematopoietic stem cells are well-known in the art, for example as disclosed in

U.S. Pat. Nos. 5,635,387 and 5,643,741, the entire disclosures of which are incorporated herein by reference. See also Campana et al. (1995) Blood 85: 1416-1434.

Preferably, harvested bone marrow is purged of tumorigenic or neoplastic cells prior to contacting the hematopoietic cells with the present compounds. Suitable purging techniques include, for example, leukopheresis of mobilized peripheral blood cells, immunoaffinity-based selection or killing of tumor cells, or the use of cytotoxic or photosensitizing agents to selectively kill tumor cells. Such techniques are well-known in the art; see, for example, Bone Marrow Processing and Purging. Part 5 (A. Gee, ed.), CRC Press, Boca Raton, Fla., 1991; Lydaki et al. (1996) J. Photochem. and Photobiol. 32: 27-32; and Gazitt et al. (1995), Blood, 86: 381-389, the entire disclosures of which are herein incorporated by reference.

The compounds of the present invention can also be used to screen for compounds which alter the binding of JLP to components of the MEKK3- MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module. Such compounds can then be used to alter JLP function in cells by preventing or encouraging operation of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module. Compounds which can potentially alter JLP activity can be identified, for example, by mixing the test compound with JLP, or fragments, derivatives, homologs or analogs thereof along with one or more signaling pathway components. The ability of the test compound to alter the association of the JLP compound with the signaling pathway component can be determined, for example, by a co-immunoprecipitation assay as described for the Max/JLP association in Example 3 below.

The alteration in JLP binding activity caused by the test compound can comprise an enhancement of JLP association with a signaling pathway component, or an inhibition of JLP association with a signaling pathway component. Another method for identifying compounds which bind to JLP (and thus potentially inhibit JLP association with signaling pathway components) is the yeast two-hybrid screening method. Briefly, nucleic acid sequences encoding various domains of JLP can be subcloned into an appropriate yeast expression vector, such as plasmid pGBKT7 (Matchmaker System 3; Clontech) to express JLP fusion proteins containing the DNA-binding domain of Gal4. The plasmids are then transfected into an appropriate yeast reporter strain, for example strain AH109, which includes the reporter genes ADE2, HIS3, lacZ and MEL1. Yeast reporter strain AH 109 is available from Clontech for the Matchmaker System 3. The yeast reporter strains are subsequently transfected with libraries of nucleic acid sequences fused to the sequences encoding the activation domain of Gal4 in pGADT7 plasmid. Preferably, cDNA or cDNA fragments from brain or skeletal muscle cDNA libraries are used to construct the pGADT7-Gal4 plasmids. Approximately 1.5 x 10 transfectants can be screened for the ability to grow on Ade+ and His+ medium, and for LacZ expression (β-galactosidase activity). Putative positive clones are tested for non-specific interaction by mating them with a yeast strain containing pGBKT7-lamin. The cDNAs from the final positive clones are rescued and sequenced to identify JLP-interacting compounds. If cDNA fragments were used to construct the pGADT7-Gal4 plasmids, the fragments rescued from the positive clones can be used as probes to screen libraries for the full-length cDNAs of JLP interacting compounds.

The invention will be illustrated with the following non-limiting examples.

Example 1 - Isolation and Characterization of Mouse JLP cDNA and protein

Isolation of Mouse JLP cDNA The cDNA coding for₍ mouse JLP was isolated by screening a λgtll expression library prepared from the mouse myeloid cell line 32Dcl3 cells with a ³²P-labeled Max probe, according to the method of Blanar and Rutter (1992), Science 256: 1014-1018, the disclosure of which is herein incorporated by reference.

The initial screen yielded 13 positive clones, which derived from mRNAs encoding for two different classes of proteins; basic/helix-loop- helix/leucine-zipper proteins such as c-Myc and Mad (which are known to associate with Max); and a second, novel class of proteins that encoded for polypeptides rich in proline residues and charged amino acids.

Analysis of Mouse JLP cDNA and Protein

The nucleotide sequence of one clone encoding the second class of proteins showed a single large open reading frame of 3.9 kb encoding a polypeptide of 1307 amino acids. The encoded protein, called JLP, was found to contain two leucine zipper domains, LZI and LZII and a C-terminal domain that showed extensive homology with C. elegans protein ZK1098.1O. JLP shares 69% homology with JSAP1 and JIP3, two proteins that act as scaffolding proteins for JNK-signaling pathway in mammalian cells.

The nucleic acid sequence of the mouse JLP cDNA clone is given in SEQ ID NO: 3, and the deduced amino acid sequence is given in SEQ ID NO: 4. Features of the mouse JLP amino acid sequence are shown in FIG. 1 A; these are: conserved domains A, B and C (boxed); the heptad repeat of leucine and homologous hydrophobic amino acids in LZI and LZII (circled); and putative SH2 and SH3 binding sites (shaded and underlined, respectively). Northern blot analysis using a JLP probe shows that it is ubiquitously expressed.

The full length mouse JLP cDNA clone of SEQ ID NO: 3 was subcloned into a modified form of pSG5 mammalian expression vector (Stratagene) by standard techniques, and used for transient transfection into COS7 cells as follows: COS7 cells growing in DMEM containing 10% fetal bovine serum were transfected with expression vector by DEAE-dextran method. Essentially, 0.6 million COS7 cells were seeded into 100mm culture dishes the day before transfection. DEAE-dextran/DNA mix was prepared containing 5 ml DMEM; 10% NuSerum (Becton-Dickinson); l-20μg vector; 200μl lOmg/ml DEAE- dextran (Sigma)/2.5 mM chloroquine (Sigma), and the cells transfected according to the method of Sussman, DJ and Milman G (1984), Mol Cell Biol 4(8), 1641-1643, the disclosure of which is herein incorporated by reference. Three hours after transfection, cells were washed with PBS and replenished with fresh growth medium. Cells were harvested 48 hours later and lysed by sonication in high stringency buffer [20 mM Tris-HCl (pH 7.5); 50 mM NaCl; ImM EDTA; 5μM DTT; 0.5% NONIDET-P40; 0.1% SDS; ImM phenylmethylsulfonyl fluoride; 2μg/ml pepstatin; 2μg/ml leupeptin; 1.9μg/ml aprotinin; lmMNa₃NO₄].

Endogenous JLP from 32Dcl3 cells was immunoprecipitated from cell lysates as follows: 32Dcl3 cells were lysed and sonicated in high stringency buffer as above for the transfected COS7 cells. The resulting lysate was kept on ice for 10 minutes, spun, and pre-cleared with pre-immune serum and Protein A Sepharose. Protein concentration was determined. 2 mg of pre-cleared lysate was used for immunoprecipitation with pre-immune serum (PI) or JLP Ν- terminal specific antibody (I) in the high stringency buffer at 4 °C for 1 hr in a rotator at 12 rpm. Protein A Sepharose was then added and the samples further mixed by rotation at 12 rpm at 4 °C for 1 hour. The immuno-complexes were washed four times with the high stringency buffer.

JLP immunoprecipitates from the 32Dcl3 cells were resolved on a 6% SDS-PAGE gel together with the lysates from COS7 cells transfected with plasmid vector alone, or plasmid vector expressing JLP, and analyzed by Western blot analysis performed with JLP Ν-terminal specific antibody I. The results, presented in FIG. 1C, show a 180 kDa protein encoded by the mouse JLP cDΝA identical to a 180 kDa endogenous JLP from 32Dcl3 cells. The discrepancy between the molecular weight of 143 kDa calculated based on the primary JLP amino acid sequence, and the apparent molecular weight of 180 kDa shown by Western blot, suggests that JLP undergoes one or more forms of post-translational modification.

Subcellular localization of JLP

The subcellular localization of endogenous JLP in SWISS3T3 cells was analyzed by indirect immunofluorescence studies as follows. Cells were permeablized with Triton X-100, followed by RΝAse A treatment and immuno- stained with JLP pre-immune antibody (PI; see above) or the JLP C-terminal specific antibody (JLP; prepared as above for PI and I antibodies), followed by a flύorescein-conjugated anti-rabbit IgG secondary antibody. The cell nuclei were stained with propidium iodide. The results revealed that JLP is a cytoplasmic protein with a punctate distribution. Isolation of Human JLP cDNA

A PCR fragment (nt399 to ntl287) of the mouse JLP cDNA was ³²P- labeled and used as a probe to screen λZapII human skeletal muscle and liver libraries (Clontech) at 37 °C in the low-stringency hybridization buffer [40 % formamide; 5X SSC; IX Denhardt's solution; sodium dextran sulfate (0.1 mg/ml); 20 mM sodium phosphate; salmon sperm DNA (0.1 mg/ml)] . Several positive clones were detected. Among the positive clones, clone #1 from the liver library and #8 from the skeletal muscle library contained the 5' and 3' end of the human JLP coding sequence, respectively. The clones were ligated at the unique overlapping Stul site to generate a cDNA sequence encoding the full length human JLP. Features of the human JLP amino acid sequence are shown in FIG. IB; these are: conserved domains A, B and C (boxed); the heptad repeat of leucine and homologous hydrophobic amino acids in LZI and LZII (circled); and putative SH2 and SH3 binding sites (shaded and underlined, respectively).

Example 2 - Association of JLP with JNK-1

JNK-1 Association with Full-Lensth Mouse JLP

To examine the association of JLP with JNK, a "pull-down" assay using S-protein agarose beads (Novagen) was performed on hemagglutinin-tagged JNKl (HA-JNKl) and S-tagged mouse JLP (JLP-S) fusion proteins co- expressed in COS7 cells. The S-tag of JLP-S is a 15 amino acid peptide ("S»Tag™"; Novagen) that binds to the 104 amino acid S-protein derived from pancreatic ribonuclease A (see Kim and Raines (1993), Protein Sci. 2: 348- 356). HA-JNKl was made by subcloning JNK-1 cDNA into the NcoI(5') and

BamHI (3') sites of the mammalian expression vector SRα3-3HA, which contains three tandem repeats of the HA coding sequence. The JNK-1 protein expressed from this plasmid contains the HA tag in its N-terminus. The SRα3 vector is described in Mol. and Cellular Biol. 8(1); 466-472 (1988), the disclosure of which is herein incorporated by reference. Briefly, the SRα3 vector contains the R segment and part of the U5 sequence of the long terminal repeat of human T-cell leukemia virus type 1, fused with the SN40 early promoter-enhancer unit, to drive the expression of HA-JΝK1. JLP-S was made by replacing the 3' end of SEQ ID NO: 3 at the unique Bglll site with a PCR fragment containing the corresponding JLP coding sequence followed by a nucleic acid sequence encoding the S-tag. The resulting JLP-S cDNA was cloned into the EcoRI (5') and the Bglll (3') sites of the constitutive expression vector pSG5puro to generate pSG5puro- JLP-S plasmid. pSG5puro was derived form pSG5 (Stratagene) by inserting a puromycin resistance cassette at the unique Nde/ site by blunt-end ligation.

COS7 cells were grown in DMEM containing 10% fetal bovine serum, penicillin (20 U/ml) and streptomycin (20 μg/ml) at 37 °C in a 5% CO₂ humidified incubator. Expression plasmids were co-transfected into COS7 cells by the DEAE-dextran method as described above for Example 1. Transfected COS7 cells expressing the HA-JNKl and JLP-S proteins were lysed according to the methods of Noguchi et al. (1999), J. Biol. Chem. 274: 32580-32587, and JLP-S precipitated from the cell lysate with S-protein agarose (Novagen), according to the manufacturer' s instructions.

The S-protein agarose precipitates were analyzed for the presence of HA-JNKl /JLP-S complexes by Western blot analysis, using mouse monoclonal anti-HA antibody F7 (sc-7392; Santa Cruz). Results of this experiment presented in FIG. 2A show that JNKl associates with JLP, as evidenced by its co-precipitation with JLP-S. When the S-protein agarose pull-down assays were performed with cell lysates expressing JLP-S alone or HA-JNKl alone, no co- precipitation was seen between JNK-1 and JLP. This indicates that there is a specific interaction between JNKl and JLP. JNK Binding Sites on Mouse JLP The amino acids involved in the association between JLP and JNKl were elucidated through a series of C-terminal deletion mutants of mouse JLP, which were all S-tagged at the C-terminal end. JLP-S deletion mutants were created by the separate digestion of the wild-type JLP cDNA EcoRI/MluI fragment isolated from pSG5puro- JLP-S plasmid with each of the following restriction enzymes: Smll, Msll, PvuII, BssSI, Spel and Bsu36I. Digestion of EcoRI and Mlul released the JLP cDNA, leaving the S-tag coding sequence in the vector. The remaining vector fragment was called pSG5puro-S vector. The cDNA fragments encoding the JLP deletion mutants were blunt ended, and ligated into the pSG5puro-S plasmid digested with EcoRI (5') and Smal or Xmal (3'). These cDNAs encoded deletion mutants that terminated at amino acid 110, 164, 374, 463, 1000 and 1165, respectively, with an S-tag at their C- terminal ends. The deletion mutants are shown schematically in FIG. 2B and are described in Table 2 below.

Table 2 - C-Terminal Deletion Mutants of JLP

COS cells were co-transfected as above with either HA-JNKl expression vector and pSG5puro-S expression vector without insert, or HA-JNKl expression vector and expression vectors encoding JLP-S or S-tagged C- terminal deletion mutants from Table 2. Cell lysates were obtained and subjected to the S-protein agarose bead pull-down assay, as above. The cell lysates were analyzed for the presence of HA-JNKl/JLP-S or HA-JNK1/S- tagged deletion mutant complexes with anti-HA antibody, as above. As shown in FIG. 2B, all of the C-terminal deletion mutants showed association with HA- JNKl. However, mutants 110 and 164 showed reduced binding to HA-JNKl, suggesting that the region spanning amino acids 1 - 374 likely contains multiple binding sites for JNKl . Additional mutants of JLP comprising fragments of the N-terminal region of JLP were constructed as follows: A PCR fragment was generated for domain II of the JLP sequence (from nt640 to ntl356), which included the Kozak sequence (GCC ACC ATG) immediately 3' of an EcoRI site, and 5' of the JLP domain II sequence. The Kozak sequence is the optimal ribosomal binding site for translation, and allows the translation of the individual domains which contain no intrinsic starting codon. The PCR fragment was digested with EcoRI and MM, then subcloned into pSG5puro-S vector digested with EcoRI/ MM.

Another PCR fragment was generated for domains II/LZII (nt640 to ntl551) and was subcloned into the BamHI and Hindlll sites of the pSG5puro-S vector. JLP-I-S and JLP-I/LZI -S are 3' deletion mutants terminated at amino acids 110 and 164, by digestion with Smll and Msll, respectively. The DNA fragment containing the coding sequence for JLP-IIΔ-S was generated from JLP cDNA sequence nt792 to ntl356 by PCR. The PCR fragment was blunt-ended, digested with MM, and subcloned into pSG5puro-S vector at the MM site and blunt BamHI site. The N-terminal region fragments are shown schematically in FIG. 2C upper panel and are described in Table 3

The positions of the amino acids comprising each fragment with respect full-length mouse JLP are shown by comparison with the structure of JLP depicted on the top of FIG. 2C. These fragments were co-expressed as S-tagged fusion proteins along with HA-JNKl in COS7 cells. S-protein agarose bead pull-down analyses were carried out on the COS7 cell lysates as described above.

SDS-PAGE analysis of the S-protein agarose bead precipitates showed that JLP-I-S (comprising JLP amino acids 1 - 110); JLP-I/LZI-S (comprising JLP amino acids 1 - 164); JLP-II-S (comprising JLP amino acids 160-398) and JLP-II/LZII-S (comprising JLP amino acids 160 - 483) associated with HA- JNKl, while JLP-IIΔ-S (deleted for JLP amino acids 160 - 209, but containing JLP amino acids 210 - 398) did not. As a loading control, the blot was also probed with an anti-S-tag antibody to show levels of JLP mutants. See FIG. 2C left panel.

These results suggest that amino acids 1 - 110 and 160 - 209 in the N- terminal region of JLP comprise the binding sites for JNKl. The results in FIG. 2C also indicate that one of these two binding sites is sufficient for JLP/JNK association.

In vivo association of endogenous JLP and JNK was also shown in COS7 cells. The COS7 cells were grown and lysed as above, and the lysates were immunoprecipitated with either a JNK-1 specific antibody or a control rabbit antibody. The immunoprecipitates were analyzed by Western blot as above, using the JLP C-terminal specific antibody described in Ex. 1. FIG. 2E shows that only the COS7 cell lysate immunoprecipitated with the JNK-1 specific antibody showed a positive reaction with the JLP C-terminal specific antibody. p38 MAP kinase Association with Full-Length Mouse JLP S-protein agarose bead pull-down experiments were also performed with p38 MAPKα tagged with FLAG (FLAG-ρ38 MAPKα) and ERK2 co-expressed with JLP-S in COS7 cells, as described above. FLAG-p38 MAPKα expression plasmid was constructed as follows: FLAG-p38 MAPKα expression plasmid was obtained from Dr. Roger Davis (Howard Hughes Medical Institute and Program of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605). This plasmid comprised a p38 MAPKα coding sequence in which the starting methionine was replaced with the FLAG- tag sequence, subcloned into pCMN5. The p38 MAPKα expression plasmid is described in Journal of Biological Chemistry 270(13):7420-7426, the entire disclosure of which is herein incorporated by reference. The endogenous ERK2 was detected in COS7 cells; no expression plasmid was involved. The COS7 cell lysates were analyzed for the presence of FLAG-p38

MAPKα/JLP-S or ERK2/JLP-S complexes with mouse anti-FLAG monoclonal antibody (F3165; Sigma) and rabbit anti-ERK2 (C-14) polyclonal antibody (sc- 154; Santa Cruz), respectively. The results shown in FIG. 2A demonstrate that JLP-S also associates with p38 MAPKα, as evidenced by co-precipitation of the two proteins. As with JΝK1, the S-protein agarose beads failed to precipitate FLAG-p38 MAPKα in the absence of JLP-S, indicating the specificity of the reaction.

JLP-S failed to interact with ERK2 (see FIG. 2A), as evidenced by a lack of co-precipitation of these proteins under conditions which demonstrated the interaction between JLP and JΝK1 or p38 MAPKα. These results indicate that JLP is distinctly different from JSAP1 and JIP3, which are known to associate only with JΝK but not with p38 MAPKα. Thus, the results show that JLP is likely a specific adaptor protein for the JΝK/p38 MAPK signaling module. p38 MAP kinase Binding Sites on Mouse JLP The amino acids of JLP that interact with p38 MAPKα were elucidated by S-protein agarose bead pull-down experiments with the JLP N-terminal region fragments from Table 3, as described above. The presence of p38 MAPKα/JLP-fragment complexes were detected in the COS7 cell lysates with anti-FLAG antibody, as above. The results presented in FIG. 2D show that JLP- I-S (comprising JLP amino acids 1 - 110); JLP-I/LZI-S (comprising JLP amino acids 1 - 164); JLP-II-S (comprising JLP amino acids 160 - 398) and JLP- II/LZII-S (comprising JLP amino acids 160 - 483) associated with p38 MAPKα, while JLP-IIΔ-S (deleted for JLP amino acids 160 - 209, but containing JLP amino acids 210 - 398) did not. These results indicate that amino acids 1-110 and 160-209 comprise the p38 MAPKα binding sites on JLP, and that either site alone is sufficient for p38 MAPKα association. The above results for JNK and p38 MAP kinase binding to JLP indicate that JLP is a scaffolding protein uniquely designed to bring JNK- and p38 MAP kinases together in one signaling module.

Example 3 - Association of JLP with Max and c-Myc

Association of Max with Full Length Mouse JLP The association of JLP with Max was demonstrated both in vitro and in vivo using co-immunoprecipitation experiments, as described below. Bacterial recombinant proteins of Max and the N-terminal region of JLP containing LZI and LZII (called "M2") were prepared as follows: The cDNA insert encoding M2, corresponding to nucleotides 114 to 1471 of the JLP cDNA (SEQ ID NO: 18) was blunt ended and subcloned into the Xmal-digested and blunt-ended pDS56-derived vector (Qiagen), while the Max cDNA was cloned into the BamHI (5') and Hindlll (3') sites of a similar vector. The resulting expression plasmids were transformed into MCI 5 bacterial host cells, which contained a /αci-expressing plasmid pDMI,l. Expression of the M2 and Max recombinant proteins, each of which contained the 6X His tag at their N- terminus, was induced in the presence of ImM IPTG. The amino acid sequence of M2 is given in SEQ ID NO: 19.

2+

The M2 and Max recombinant proteins were purified using Ni -NTA

I agarose resin (Qiagen) under denaturing condition using 6M guanidine-HCl.

Briefly, the induced cells were resuspended in Buffer A (50mM sodium phosphate; 6M guanidine-HCl, pH 8.0), and lysed at 4 °C overnight. The lysate was spun at 15K for 20 min., and the supernatant was passed through a column containing Ni2⁺-NTA agarose resin, equilibrated with Buffer A (pH 8.0). The column was then washed with Buffer A (pH 8.0), followed by Buffer A (pH 6.0). Finally, the column was eluted with Buffer A (pH 5.0). The eluate was dialyzed stepwise in the following buffers: Buffer B (1.0M guanidine-HCl; 50mM sodium phosphate; 1 mM DTT, pH 7.5), Buffer C (0.1M guanidine-HCl; 50mM sodium phosphate; ImM DTT, pH 7.5), and Buffer D (20mM HEPES; 20% glycerol; 1 mM DTT, pH 7.5). The purified recombinant Max and M2 proteins were mixed and immunoprecipitated either with the pre-immune serum or rabbit anti-Max (C17) polyclonal antibody (scl97; Santa Cruz), according to the procedure described on pg. 15 above for the in vitro association of compounds with MEKK3-MKK4- JNK/p38 MAPK/c-Myc/MAX signaling module components.

Immunocomplexes were resolved by SDS-PAGE followed by Western blot analysis with the JLP N-terminal specific antibody (see above). FIG. 3A shows that M2 was readily immunoprecipitated from the mixture by Max- specific antibodies, but not by the pre-immune serum. These results indicate that the N-terminal domain of JLP containing the two leucine-zippers associates with Max. The precipitation of M2 was not due to the non-specific binding of the Max antibody to M2, since these antibodies could not precipitate M2 in the absence of Max (FIG. 3 A).

Max Binding Site on JLP To determine the domains responsible for JLP-Max association, co- immunoprecipitation experiments were performed as described above, using Max mutants previously described by Reddy et al. (1992), Oncogene 7: 2085- 2092, the entire disclosure of which is herein incorporated by reference, in which the basic, helix-loop-helix and leucine-zipper domains of Max were mutated (see FIG. 3B). Recombinant wild type and mutant forms of Max prepared as in Reddy, supra, were incubated with M2, followed by immunoprecipitation with Max antibody (Max-Ab, see above), and analyzed by SDS-PAGE. As a loading control, the gel was stained with Coomassie blue to show the levels of wild type and mutant Max proteins. FIG. 3B shows that wild type Max and Max mutants with mutations in the basic, the first helix and the second helix domains co-immunoprecipitated with M2. The Max mutant containing alterations in the leucine-zipper domain did not co-immunoprecipitate with M2. This indicates that the leucine-zipper domain of Max is essential for its interaction with M2. The amount of M2 co-immunoprecipitated with mutant protein of Max containing alterations in the first helix and the second helix domains was higher than that seen with the wild type Max and the Max mutant containing alterations in the basic domain. This is likely because mutations in Max helix-loop-helix domain abolish Max homodimer formation, resulting in greater availability of Max monomer for association with M2.

To determine the domain of M2 that is responsible for its association with Max, an experiment was carried out using a mutant form of JLP called

"M2LZI," which has all of the leucine residues of the first leucine zipper (LZI) from JLP mutated to alanine. M2LZI is encoded by the nucleic acid of SEQ ID

NO: 20, and the amino acid sequence of M2LZI is given in SEQ ID NO: 21.

Recombinant M2LZI was prepared as above for M2, and immunoprecipitation experiments showed that this mutant protein fails to associate with Max (FIG. 3C). These results indicate that LZI of M2 is involved in Max binding to JLP, and the second leucine-zipper does not participate in this interaction. This was confirmed by immunoprecipitation experiments with a second M2 mutant called M2LZII, in which the leucines of LZII were mutated to alanine. M2LZII is encoded by the nucleic acid of SEQ ID NO: 22, and the amino acid sequence is given in SEQ ID NO: 23. M2LZII did not show a reduced association with Max.

In Vivo Association Max and JLP

To demonstrate in vivo association between Max and JLP, JLP-S (see Example 2, above) and wild-type Max (prepared as in Reddy et al., supra) were co-expressed in COS7 cells and subjected to S-protein agarose bead pull-down assay as described above in Example 2 above. The precipitate was examined for the presence of Max/JLP complexes via Western blot analysis using Max-Ab (see above) and anti-S-tag antibody (Novagen). The results, shown in FIG. 3D, demonstrate that Max readily associates with JLP in vivo.

Effect of Max/JLP Association on Max/Max Homodimerization

Electrophoretic mobility shift assays with recombinant c-Myc, Max and M2 proteins were performed using the ³²P-labeled double-stranded oligonucleotide, CM1, which contained the E-box sequence (CACGTG). Bacterial recombinant c-Myc and Max proteins described previously in Reddy et al., supra, were mixed as indicated therein with the labeled probe in the presence or absence of the bacterial recombinant protein M2. The DNA/protein complexes were resolved in a native non-denaturing polyacrylamide gel (4%). After electrophoresis, the gel was dried and subjected to autoradiography.

The results, shown in FIG. 3E, showed that the CM1 probe readily bound to Max/Max homodimers and c-Myc/Max heterodimers, as evidenced by the appearance of shifted bands of different mobilities. When recombinant c-

Myc alone or M2 alone was used, there was no detectable binding of the probe to either protein.

Addition of M2 to the reaction mixture containing recombinant Max and the labeled probe disrupted Max/Max homodimer formation. However, when M2 was added to the reaction mixture containing recombinant c-Myc and Max, little or no effect was observed on the formation of c-Myc/Max heterodimers (see FIG. 3E). These results indicate that JLP has a high affinity for Max/Max homodimers, and its interaction with Max results in the dissociation of these homodimers. However, the affinity of JLP for Max appears weaker than that of c-Myc, as evidenced by the inability of JLP to dissociate c-Myc/Max heterodimers.

Association ofc-Mvc with Full Length JLP

COS7 cells were transiently transfected to express JLP-S and wild type c-Myc, as described above in Example 2. Plasmid vector expressing wild-type c-Myc was prepared by subcloning the cDNA containing the coding sequence of human c-Myc into the EcoRI site of pSG5puro vector. Precipitates from COS7 cell lysates were examined for the presence of c-Myc/JLP complexes via

Western blot analysis using rabbit anti-c-Myc (N262) polyclonal antibody (sc-

764; Santa Cruz) or antibodies specific for the S-tag (Novagen). The results presented in Fig. 4A show that c-Myc readily associated with JLP. c-Myc Binding Sites on JLP

To determine the domain of JLP involved in its association with c-Myc, vectors that expressing the different N-terminal domains of JLP (see Table 3 above) were transfected into COS7 cells along with a c-Myc expression vector as above in Example 2. The transfected COS7 cells were lysed, the cell lysates precipitated with S-protein agarose beads as described above in Example 2. SDS-PAGE analysis of the precipitates with c-Myc specific antibodies showed that the JLP N-terminal fragments containing the region spanning amino acids 1 - 110 (JLP-I-S; SEQ ID NO: 12) and 160 - 398 (JLP-II-S; SEQ ID NO: 16) associate with c-Myc, while the shorter polypeptide spanning between amino acid position 210 - 398 (JLP-IIΔ-S; SEQ ID NO: 17) did not. See FIG.4B. These results indicate that amino acids 1 - 110 at the N-terminus of JLP, or amino acids 160 - 209 located between the two leucine-zipper domains, are necessary for the association of JLP with c-Myc. Each region alone appears sufficient for c-Myc/JLP binding. Neither of the two leucine zipper domains of JLP appear to play a role in binding of JLP to c-Myc. FIGS. 2C and 4B show that c-Myc and JNKl binding involve the same regions of JLP.

Example 4 - Association of JLP and MKK4 and MEKK3

COS7 cells were transiently transfected to express GST-tagged MKK4, HA-tagged MEKK3 and MKK3 with or without JLP-S, according to the method of Noguchi et al. (1999), J. Biol. Chem. 2_14. 32580-32587, the entire disclosure of which is herein incorporated by reference. Cell lysates from the transfected cells were subjected to a pull-down assay using S-protein agarose beads as described above in Example 2. The precipitates and the lysates were analyzed with rabbit anti S-probe antibody (against S-tag, K14/sc802), mouse anti-HA antibody (F7/sc7392)and rabbit anti-GST antibody (Z5/sc459), all from Santa Cruz. As shown in FIG. 5 A, JLP associates only with MKK4 and MEKK3, but not with MKK3.

In a further experiment, COS7 cells were transiently transfected as above to express HA-JNKl and GST-MKK4, with or without JLP-S. Cell lysates from the transfected cells were subjected to a pull-down assay using S-protein agarose or glutathione-Sepharose beads as in Example 2, above. The cell lysates and corresponding precipitates were analyzed by Western blot with HA or GST specific antibodies. As shown in FIG. 5B, show that GST-MKK4 could "pull-down" HA-JNKl only when cotransfected with JLP, indicating that JLP can function as a scaffolding protein that can simultaneously bind JNK and MKK4 (which is an upstream kinase of JNK).

The data shown in FIGS. 5A and 5B, taken together with the data of Examples 2 and 3, strongly indicate that JLP is a scaffolding protein that brings JNKl and p38 MAPKα in close proximity to the upstream kinases MKK4 and MEKK3 to form a functional MEKK3-MKK4-JNK/p38 MAPK-c-Myc/Max signaling module.

Example 5 - Modulation of Apoptotic Response with JLP

JLP was shown to activate cell survival pathways in cells undergoing an environmentally-induced stress response. Typically, cells undergoing such a stress response experience apoptosis mediated by the JNK/p38 MAP kinase signaling pathway.

Preparation of Rat 1 A Cells Expressing JLP

Clonal lines of RatlA cells transfected with IPTG-inducible JLP expression vector or expression vector without JLP were prepared as follows. Mouse JLP cDNA (SEQ ID NO: 3) was subcloned into plasmid pOPI3 (Stratagene) to form plasmid pOPI3-JLP. RatlA cells were transfected sequentially with the Lacl-expressing plasmid p3'SS and pOPI3-JLP by the calcium phosphate method. Briefly, RatlA cells were seeded into 100 mm culture dishes at a density of 0.4 x 10⁶ cells/dish the day before transfection. On the day of transfection, 10 μg Xmn/-digested p3'SS was resuspended in 450μl water and 50 μl 2.5M CaCl₂. The DNA solution was added dropwise with mixing to 0.5ml of 2X HEPES buffer (50mM HEPES; 50mM Na₂HPO₄; 0.5M NaCl, pH 7.45). The resulting solution was added directly to the culture medium in the dish. Cells were washed next day and replenished with fresh culture medium. Hygromycin (100 mg/ml) was added to the medium on the next day. Clonal cells expressing Lad were selected for subsequent transfection. 5μg of Nhe/-digested pOPI3 or pOPI3-JLP was used to transfect the Lacl- expressing RatlA clonal cells using the same method, except that G418 (0.4 mg/ml) was used for selection. Clonal cells expressing high level of JLP in the presence of 5mM IPTG were used for the experiment.

Cell lysates were prepared as described in Noguchi et al., supra. Western blot analysis of the cell lysates derived from pOPI3 -vector transfected cells (N-RatlA) and pOPI3-JLP transfected cells (JLP-RatlA) showed considerably higher levels of JLP expression in JLP-RatlA cells as compared to N-Ratl A cells, even in the absence of IPTG (FIG. 6A). Addition of IPTG to these cells resulted in a considerable increase in the levels of JLP in JLP-RatlA cell line (FIG. 6A).

Activation of Cell Survival Pathway by Expression of JLP N-Ratl A and JLP-RatlA cells were then exposed to a lethal dose of UN (254 nm for 5 min) to induce apoptosis, in the presence and absence of IPTG. The viability of the cells was monitored by trypan blue exclusion at 0, 12, 23 and 48 hr., as shown in FIG. 6B. The mean + SD from three independent replicates was used to plot the total cell viability in FIG. 6C.

The N-RatlA cells exhibited high rate of apoptosis which was time- dependent; approximately 75% cell death was observed at 48 hrs. following UN-exposure. In contrast, the JLP-RatlA cells did not undergo significant cell death upon UN exposure; approximately 80% of the cells remained viable after 48 hrs following UV exposure. The protection was limited, as the JLP-RatlA cells eventually died by 96 hr after the UV treatment. Interestingly, the JLP- RatlA cells showed "leaky" expression of JLP, as protection against UV- induced stress apoptosis by JLP was also seen in JLP-RatlA cells not treated with IPTG (see FIG. 6C, which shows that the viability of uninduced Ratl A- JLP cells is about 85% of those induced with IPTG). This indicates that even low level expression of JLP can activate cell survival pathways in cells undergoing a JΝK/p38 MAP kinase-mediated stress response.

Example 6 - Modulation of JLP Phosphorylation by JLP

To demonstrate the role of JLP in JNK activation, NIH-3T3 cells were cultured according to standard techniques and were transiently transfected with expression plasmids for JNK1-S, FLAG-p38 MAPKα, HA-MKK4 and HA- MEKK3. The cells were also transfected with expression plasmids for HA- tagged wild-type mouse JLP, a dominant negative mutant of JLP lacking the JNK binding domains (ΔJBD) or an empty vector, in the presence or absence of a dominant positive mutant of MEKK1 (ΔMEKK). To construct ΔJBD, the JNK binding domains from amino acids 1-107 and 197-209 were deleted from wild type JLP. One day after transfection, cells were lysed and the JNK1-S was precipitated from the lysates by S-protein agarose. The precipitates were analyzed by Western blot using a phospho-JNKl specific antibody. Previous studies demonstrated that ΔMEKK activates JNK and p38MAPK pathways in a constitutive manner (Ito M et al. (1999), Mol Cell. Biol. 19: 7539-7548). FIG. 7 shows that JNK activation in the presence of ΔMEKK was enhanced by coexpression of JLP and abolished by coexpression of ΔJBD. These data indicate that JLP assembles components of the JNK pathway for further activation.

All documents referred to herein are incorporated by reference. While the present invention has been described in connection with the preferred embodiments of the various figures, it is to be understood that other similar embodiments may be used or modifications and additions made to the described embodiments for performing the same function of the present invention without deviating therefrom. Therefore, the present invention should not be limited to any single embodiment, but rather should be construed in breadth and scope in accordance with the recitation of the appended claims.

Claims

We claim:

1. An isolated nucleic acid sequence comprising:

(a) SEQ ID NO: 1;

(b) a nucleic acid sequence complementary to (a);

(c) a nucleic acid having at least 70% sequence identity with (a) or (b); or

(d) a nucleic acid sequence that hybridizes to (a) or (b) in 7% sodium dodecyl sulfate, 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 2XSSC, 0.1% sodium dodecyl sulfate at 50 °C.

2. The nucleic acid of claim 1, wherein (d) comprises a nucleic acid sequence of at least 18 sequential nucleotides of (a) or (b).

3. The nucleic acid of claim 1, wherein (c) or (d) encodes a protein that:

(i) associates in vivo or in vitro with at least one component of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module; or

(ii) comprises at least one epitope of human JLP.

4. The nucleic acid of claim 1, wherein (c) has at least 75% sequence identity with (a) or (b).

5. The nucleic acid of claim 1, wherein (c) has at least 80% sequence identity with (a) or (b).

6. The nucleic acid of claim 1, wherein (c) has at least 90% sequence identity with (a) or (b).

7. The nucleic acid of claim 1, wherein (c) has at least 95% sequence identity with (a) or (b).

8. The nucleic acid of claim 1, wherein (c) has at least 98% sequence identity with (a) or (b).

9. A nucleic acid encoding the peptide of SEQ ID NO: 2.

10. An isolated nucleic acid sequence comprising:

(a) SEQ ID NO: 3;

(b) a nucleic acid sequence complementary to (a);

(c) a nucleic acid having at least 70% sequence identity with (a) or (b); or

(d) a nucleic acid sequence that hybridizes to (a) or (b) in 7% sodium dodecyl sulfate, 0.5 M NaPO₄, 1 mM EDTA at 50 °C with washing in 2XSSC, 0.1% sodium dodecyl sulfate at 50 °C.

11. The nucleic acid of claim 10, wherein (d) comprises a nucleic acid sequence of at least 18 sequential nucleotides of (a) or (b).

12. The nucleic acid of claim 10, wherein (b) or (c) encodes a protein that:

(i) associates in vivo or in vitro with at least one component of the MEKK3-MKK4-JNK p38 MAPK/c-Myc/MAX signaling module; or

(ii) comprises at least one epitope of mouse JLP.

13. The nucleic acid of claim 10, wherein (c) has at least 75% sequence identity with (a) or (b).

14. The nucleic acid of claim 10, wherein (c) has at least 80% sequence identity with (a) or (b).

15. The nucleic acid of claim 10, wherein (c) has at least 90% sequence identity with (a) or (b).

16. The nucleic acid of claim 10, wherein (c) has at least 95% sequence identity with (a) or (b).

17. The nucleic acid of claim 10, wherein (c) has at least 98% sequence identity with (a) or (b).

18. A nucleic acid encoding the peptide of SEQ ID NO: 4.

19. A compound comprising SEQ ID NO: 2, or a biologically active fragment, derivative, homolog or analog thereof.

20. The compound of claim 19, further comprising a modification allowing entry of the protein into a cell when the protein is added exogenously to the cell.

21. The compound of claim 20, wherein the modification comprises a protein transduction domain.

22. A compound comprising SEQ ID NO: 4, or a biologically active fragment, derivative, homolog or analog thereof.

23. The compound of claim 22, further comprising a modification allowing entry of the protein into a cell when the protein is added exogenously to the cell.

24. The compound of claim 23, wherein the modification comprises a protein transduction domain.

25. The compound of claim 22, wherein the fragment is selected from the group consisting of SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 19.

26. The compound of claim 22, wherein the derivative comprises SEQ ID NO: 21 or SEQ ID NO: 23.

27. An antibody that specifically binds to an epitope on the compound of claim 19.

28. The antibody of claim 27 which is a polyclonal antibody.

29. The antibody of claim 27 which is a monoclonal antibody.

30. A hybridoma producing the monoclonal antibody of claim 30.

31. An antibody that specifically binds to an epitope on the compound of claim 22.

32. The antibody of claim 31 which is a polyclonal antibody.

33. The antibody of claim 31 which is a monoclonal antibody.

34. A hybridoma producing the monoclonal antibody of claim 33.

35. A method of modulating the apoptotic response in a cell exposed to, or in danger of being exposed to, an apoptosis-inducing environmental stress comprising contacting the cell with an effective amount of a compound comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a biologically active fragment, derivative, homolog or analog thereof, such that the compound is introduced into the cell so that the cell does not undergo apoptosis in response to the environmental stress.

36. The method of claim 35, wherein the cell does not undergo the stress-induced apoptosis for at least 12 hours from being exposed to the apoptosis-inducing environmental stress.

37. The method of claim 35, wherein the cell does not undergo the stress-induced apoptosis for at least 24 hours from being exposed to the apoptosis-inducing environmental stress.

38. The method of claim 35, wherein the cell does not undergo the stress-induced apoptosis for at least 48 hours from being exposed to the apoptosis-inducing environmental stress.

39. The method of claim 35, wherein the apoptosis-inducing stress is selected from the group consisting of ionizing radiation and anti-cancer drugs.

40. The method of claim 35, wherein said contact comprises transfecting the cell with a nucleic acid encoding the compound, such that the compound is expressed in the cell.

41. The method of claim 40, wherein the nucleic acid comprises a plasmid vector comprising SEQ ID NO: 1 or SEQ ID NO: 3.

42. The method of claim 35, wherein the compound further comprises a modification such that the compound enters the cell when administered exogenously.

43. The method of claim 42, wherein the modification comprises association of the compound with a protein transduction domain.

44. A method of protecting hematopoietic stem cells from apoptotic death induced by anti-cancer radiation or chemotherapy treatments, comprising the steps of:

(1) removing a portion of bone marrow containing hematopoietic stem cells from a subject prior to administering anti-cancer therapy;

(2) maintaining the hematopoietic stem cells in culture;

(3) contacting the hematopoietic stem cells with an effective amount of a compound comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a biologically active fragment, derivative, homolog or analog thereof, such that the hematopoietic stem cells do not undergo apoptosis when subjected to apoptosis-inducing environmental stress from anti-cancer radiation or chemotherapy treatments;

(4) reintroducing the cells into the subject; and

(5) optionally administering at least one further anti- cancer treatment to the subject.

45. The method of claim 44, wherein the hematopoietic stem cells are stably transfected with a nucleic acid comprising a plasmid vector comprising SEQ ID NO: 1 or SEQ ID NO: 3.

46. A method of identifying a compound which alters the association of JLP with a component of the MEKK3-MKK4-JNK/p38 MAPK/c-Myc/MAX signaling module, comprising the steps of:

(1) providing a JLP compound comprising SEQ ID NO: 2 or SEQ ID NO: 4, or a biologically active fragment, derivative, homolog or analog thereof;

(2) contacting the JLP compound with a signaling pathway component and a test compound; and

(3) determining whether the test compound causes an alteration in in vivo or in vitro association of the signaling pathway component with the JLP compound.

47. The method of claim 46, wherein determining whether the test compound caused an alteration in in vivo or in vitro association of the signaling pathway component with the JLP compound comprises a co- immunoprecipitation assay.

48. The method of claim 46, wherein the alteration comprises enhancement of JLP association with the signaling pathway component.

49. The method of claim 46, wherein the alteration comprises inhibition of JLP association with the signaling pathway component.