WO1999041385A1 - Proteines mekk1 et leurs fragments destines a etre utilises dans la regulation d'une apoptose - Google Patents

Proteines mekk1 et leurs fragments destines a etre utilises dans la regulation d'une apoptose Download PDF

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Publication number
WO1999041385A1
WO1999041385A1 PCT/US1999/002974 US9902974W WO9941385A1 WO 1999041385 A1 WO1999041385 A1 WO 1999041385A1 US 9902974 W US9902974 W US 9902974W WO 9941385 A1 WO9941385 A1 WO 9941385A1
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mekkl
nucleic acid
protein
seq
acid molecule
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PCT/US1999/002974
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English (en)
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Gary L. Johnson
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Cadus Pharmaceutical Corporation
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Priority to AU32895/99A priority Critical patent/AU3289599A/en
Publication of WO1999041385A1 publication Critical patent/WO1999041385A1/fr
Priority to US11/004,057 priority patent/US20050244846A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to isolated nucleic acid molecules encoding MEKKl proteins, substantially pure MEKKl proteins, and products and methods for regulating apoptosis in cells.
  • Mitogen-activated protein kinase (Mitogen- Activated Protein Kinases, also called extracellular signal-regulated kinases or ERKs) -are rapidly activated in response to ligand binding by both growth factor receptors that are tyrosine kinases and receptors that are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins).
  • MAPKs integrate multiple intracellular signals transmitted by various second messengers via a mechanism which involves the phosphorylation and regulation of the activity of enzymes and transcription factors including the EGF receptor, Rsk 90, phospholipase A 2 , c-Myc, c-Jun and Elk-1/TCF.
  • MAPKs are in turn phosphorylated and regulated by proteins called MEKs (MAPK Kinase or ERK Kinase) or MKK (MAP Kinase kinase).
  • MEKs MAPK Kinase or ERK Kinase
  • MKK MAP Kinase kinase
  • the MEKs phosphorylate MAPKs on both tyrosine and threonine residues which results in activation of MAPKs.
  • MEKs are likewise phosphorylated and regulated by one of two distinct classes of mammalian serine-threonine protein kinase, the Rafs or the MEKKs (MEK Kinases).
  • Certain biological functions are tightly regulated by signal transduction pathways within cells.
  • Signal transduction pathways maintain the balanced steady state functioning of a cell.
  • Disease states can arise when signal transduction in a cell breaks down, thereby removing the tight control that typically exists over cellular functions. For example, tumors develop when regulation of cell growth is disrupted, enabling a clone of cells to expand indefinitely.
  • signal transduction networks regulate a multitude of cellular functions depending upon the cell type, a wide variety of diseases can result from abnormalities in such networks. Devastating diseases such as cancer, autoimmune diseases, allergic reactions, - 2 -
  • inflammation, neurological disorders and hormone-related diseases can result from abnormal signal transduction.
  • the present invention is based, at least in part, on the identification of MEKKl protein and nucleic acid molecules, in particular, human MEKKl molecules, as well as bioactive fragments of MEKKl molecules useful in regulating cellular apoptosis.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:3.
  • the sequence of SEQ ID NO:3 corresponds to a murine MEKKl cDNA.
  • the predicted amino acid sequence of murine MEKKl is set forth as SEQ ID NO 4.
  • This cDNA comprises sequences encoding the murine MEKKl protein (i.e., "the coding region”, from nucleotides 15-4496), as well as 5' untranslated sequences (nucleotides 1-14) and 3' untranslated sequences (nucleotides 4497-5253).
  • the predicted amino acid sequence of murine MEKKl is set forth as SEQ ID NO 4.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:5.
  • the sequence of SEQ ID NO:5 corresponds to a human MEKKl cDNA.
  • This cDNA comprises sequences encoding human MEKKl protein (i.e., a "coding region", from nucleotides 3-3911).
  • the predicted amino acid sequence of human MEKKl is set forth as SEQ ID NO 5.
  • this invention provides isolated nucleic acid molecules encoding MEKKl proteins or biologically active portions or fragments thereof (e.g., apoptotic portions or fragments), as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of MEKKl -encoding nucleic acids.
  • a MEKKl nucleic acid molecule is 90% homologous to the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:5, or complement thereof.
  • an isolated MEKK nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:3, or a complement thereof.
  • a MEKK nucleic acid molecule comprises nucleotides 15-4496 of SEQ ID NO:3. In - 3 -
  • an isolated MEKK nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:5.
  • a MEKKl nucleic acid molecule comprises nucleotides 3-3911 of SEQ ID NO:5.
  • a MEKKl nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence substantially homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
  • a MEKKl nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
  • a MEKKl nucleic acid molecule is a naturally occurring nucleotide sequence (e.g., a naturally- occurring human or murine nucleotide sequence).
  • an isolated nucleic acid molecule which specifically detect MEKKl nucleic acid molecules relative to nucleic acid molecules encoding non-MEKKl proteins.
  • an isolated nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:5, or a complement thereof.
  • an isolated nucleic acid molecule hybridizes to about nucleotides 1-2400 of SEQ ID NO:3.
  • an isolated nucleic acid molecule is at least 500 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:5, or a complement thereof
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a MEKKl nucleic acid.
  • Another aspect of the invention provides a vector comprising a MEKKl nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention also provides a method for producing a MEKKl protein by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a MEKKl protein is produced.
  • Another aspect of this invention features isolated or recombinant MEKKl proteins and polypeptides.
  • an isolated protein includes a biologically active portion of a MEKKl protein (e.g., an apoptotic portion).
  • an isolated MEKKl protein has an amino acid sequence substantially homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
  • a MEKKl protein has an amino acid sequence at least about 90% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
  • a MEKKl protein has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
  • Another embodiment of the invention features an isolated MEKKl protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 90% homologous to a nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, or a complement thereof.
  • This invention further features an isolated MEKKl protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, or a complement thereof.
  • the MEKKl proteins of the present invention can be operatively linked to a non-MEKKl polypeptide (e.g., heterologous amino acid sequences) to form MEKKl fusion proteins.
  • the invention further features antibodies that specifically bind MEKKl proteins, such as monoclonal or polyclonal antibodies.
  • the MEKKl proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of a MEKKl protein in a sample (e.g., biological sample) by contacting the sample with a compound which selectively binds to the protein and determining whether the compound binds to the protein in the sample to thereby detect the presence of a MEKKl protein in the sample.
  • a sample e.g., biological sample
  • the present invention provides a method for detecting the presence of a MEKKl nucleic acid molecule in a sample (e.g., biological sample) by contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule and determining whether the probe or primer binds to a nucleic acid molecule in the sample to thereby detect the presence of a MEKKl nucleic acid molecule in the sample.
  • a sample e.g., biological sample
  • the present invention provides a method for detecting the presence of MEKKl activity in a biological sample by contacting the biological sample with an agent capable of detecting MEKKl activity such that the presence of MEKKl activity is detected in the biological sample.
  • the invention provides a method for modulating MEKKl activity comprising contacting the cell with an agent that modulates MEKKl activity such that MEKKl activity in the cell is modulated.
  • the agent inhibits MEKKl activity.
  • the agent stimulates MEKKl activity.
  • the agent is an antibody that specifically binds to a MEKKl protein.
  • the agent modulates expression of MEKKl by modulating transcription of a MEKKl gene or translation of a MEKKl mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a MEKKl mRNA or a MEKKl gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant MEKKl protein or nucleic acid expression or activity by administering an agent which is a MEKKl modulator to the subject.
  • the MEKKl modulator is a MEKKl protein.
  • the MEKKl modulator is a MEKKl nucleic acid molecule.
  • the MEKKl modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant MEKKl protein or nucleic acid expression is a developmental, differentiative, proliferative disorder, an immunological disorder, or cell death.
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a MEKKl protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of a MEKKl protein, wherein a wild-type form of said gene encodes an protein with a MEKKl activity.
  • the present invention also includes methods to use MEKKl proteins to regulate apoptosis.
  • the invention provides active fragments of MEKKl proteins that are generated upon cleavage of MEKKl with a caspase protease. These active fragments are capable of stimulating apoptosis.
  • the invention provides protease- resistant forms of MEKKl proteins, that are resistant to cleavage by caspase proteases and that are capable of inhibiting apoptosis. Still further, the invention provides methods for generating an active fragment of MEKKl, methods of identifying modulators of the apoptotic activity of an active fragment of MEKKl and methods of identifying modulators of caspase-mediated cleavage of MEKKl.
  • MEK kinase 1 a 196 kDa protein kinase
  • Cleavage of mouse MEKKl at Asp 874 generates a 91 kDa kinase fragment and a 113 kDa NH2-terminal fragment.
  • the kinase fragment of MEKKl induces apoptosis.
  • Cleavage of MEKKl and apoptosis are inhibited by p35 and CrmA, viral inhibitors of the ICE/FLICE proteases that commit cells to apoptosis.
  • MEKKl undergoes a phosphorylation-dependent activation followed by its proteolysis.
  • MEKKl activation and cleavage occurs in response to genotoxic agents and the activated kinase fragment functions to commit cells to apoptosis.
  • this invention defines MEKKl as a protease substrate that when activated and cleaved stimulates an apoptotic response.
  • the proteolytic cleavage of MEKKl defines the mechanism to generate a protein kinase whose activity is sufficient to induce apoptosis.
  • the finding that the activation and cleavage of MEKKl occurs in response to genotoxic agents is particularly important. It has been found that expression of MEKKl is capable of killing by apoptosis cells that have both p53 alleles mutated. Hence, the activation and cleavage of MEKKl is an apoptotic pathway that does not require a functional p53 and stimulation of these events could enhance the killing of many different tumors. Manipulating the activation of MEKKl and its cleavage by proteases, with the use of drugs for example, could increase the killing of tumor cells to genotoxic agents. Consistent with this hypothesis is the finding that low level expression of MEKKl potentiated the apoptotic response to low doses of UV irradiation and cisplatin.
  • One aspect of the present invention pertains to active fragments of MEKKl proteins (i.e., fragments of MEKKl proteins that retain apoptotic activity).
  • active fragments can be generated naturally by cleavage of MEKKl by a caspase protease.
  • an apoptotic fragment of murine MEKKl can be generated by caspase after a cleavage site found at amino acids 871-874 of SEQ ID NO:4.
  • an apoptotic fragment of human MEKKl can be generated by caspase after a cleavage site found at amino acids 681-684 of SEQ ID NO:6.
  • the active fragments of the invention can be prepared by recombinant DNA technology, using standard methodologies.
  • the invention provides an isolated active fragment of an MEKKl protein consisting of an amino acid sequence having at least 75% homology to an amino acid sequence consisting of about amino acids 875-1493 of SEQ ID NO:4, wherein said active fragment mediates apoptosis.
  • the active fragment consists of an amino acid sequence having at least 85% homology to an amino acid sequence consisting of about amino acids 875-1493 of SEQ ID NO:4. More preferably, the active fragment consists of an amino acid sequence having at least 95% homology to an amino acid sequence consisting of about amino acids 875-1493 of SEQ ID NO:4.
  • the active fragment is a mouse MEKKl active fragment.
  • the active fragment is a human MEKKl active fragment.
  • the active fragment is a rat MEKKl active fragment.
  • the active fragment can consist of, for example, about amino acids 875-1493 of SEQ ID NO:4. - 7 -
  • the active fragment consists of amino acids 875-1493 of SEQ ID NO:4.
  • the active fragment can consist of about amino acids 685-1303 of SEQ ID NO:6.
  • the active fragment consists of amino acids 685-1303 of SEQ ID NO:4.
  • protease-resistant forms of MEKKl proteins can be generated by mutation of the caspase cleavage site in an MEKKl protein (e.g., a cleavage site corresponding to amino acids 871-874 of SEQ ID NO:4 or amino acids 681-684 of SEQ ID NO:6) such that the site cannot be cleaved by the caspase.
  • an MEKKl protein e.g., a cleavage site corresponding to amino acids 871-874 of SEQ ID NO:4 or amino acids 681-684 of SEQ ID NO:6
  • at least the Asp residue at 871 and/or 874 of SEQ ID NO:4 is mutated.
  • at least the Asp residue at 681 and/or 684 of SEQ ID NO:6 is mutated.
  • one or more of the amino acids corresponding to 871-874 of SEQ ID NO:4 or to 681-684 of SEQ ID NO:6 can be mutated to, for example, alanine residues. Alternatively, said residue can be mutated to glutamine.
  • the invention provides an isolated protease-resistant MEKKl protein comprising an amino acid sequence having at least 75% homology to the amino acid sequence of SEQ ID NO:4, wherein at least one amino acid equivalent to amino acids 871-874 of SEQ ID NO:4 is substituted such that the MEKKl protein is resistant to proteolysis by a caspase.
  • the protease-resistant MEKKl protein has at least 85%o homology to the amino acid sequence of SEQ ID NO:4.
  • the protease-resistant MEKKl protein has at least 95% homology to the amino acid sequence of SEQ ID NO:4.
  • the protease-resistant MEKKl protein is a mouse MEKKl protein.
  • the protease-resistant MEKKl protein is a human MEKKl protein.
  • the protease-resistant MEKKl protein is a rat MEKKl protein.
  • the invention further provides isolated nucleic acid molecules that encode the MEKKl active fragments of the invention.
  • the invention provides an isolated nucleic acid molecule consisting of a nucleotide sequence having at least 75%> homology to a nucleotide sequence consisting of about nucleotides 2637-4493 of SEQ ID NO:3, wherein said nucleic acid molecule encodes an active fragment of MEKKl that mediates apoptosis.
  • the nucleic acid molecule consists of a nucleotide sequence having at least 85% homology to a nucleotide sequence consisting of about nucleotides 2637-4493 of SEQ ID NO:3.
  • the nucleic acid molecule consists of a nucleotide sequence having at least 95% homology to a nucleotide sequence consisting of about nucleotides 2637-4493 of SEQ ID NO:3.
  • the nucleic acid molecule encodes an active fragment of mouse MEKKl .
  • the nucleic acid molecule encodes an active fragment of human MEKKl .
  • the nucleic acid molecule encodes an active fragment of rat MEKKl .
  • the nucleic acid molecule comprises at least about nucleotides 2637-4493 of SEQ ID NO:3, or a nucleotide sequence that, due to the degeneracy of the genetic code, encodes the same amino acid sequence as about nucleotides 2637-4493 of SEQ ID NO:3.
  • the nucleic acid molecule comprises at least about nucleotides 2052-3908 of SEQ ID NO:5, or a nucleotide sequence that, due to the degeneracy of the genetic code, encodes the same amino acid sequence as nucleotides 2052-3908 of SEQ ID NO:5.
  • the invention also provides isolated nucleic acid molecules encoding the protease-resistant forms of MEKKl of the invention.
  • the invention provides an isolated nucleic acid molecule encoding a protease-resistant MEKKl protein, wherein the protease resistant MEKKl protein comprises an amino acid sequence having at least 75% homology to the amino acid sequence of SEQ ID NO:4 and at least one codon of the nucleic acid molecule encoding an amino acid equivalent to at least one of amino acids 871-874 of SEQ ID NO:4 is mutated such the encoded MEKKl protein is resistant to proteolysis by a caspase after an amino acid equivalent to amino acid 874 of SEQ ID NO:4.
  • the MEKKl protein comprises an amino acid sequence having at least 85% homology to the amino acid sequence of SEQ ID NO:4. More preferably, the MEKKl protein comprises an amino acid sequence having at least 95% homology to the amino acid sequence of SEQ ID NO:4.
  • the nucleic acid encodes a protease-resistant mouse MEKKl protein. In another embodiment, the nucleic acid encodes a protease-resistant human MEKKl protein. In yet another embodiment, the nucleic acid molecule encodes a protease- resistant rat MEKKl protein.
  • the nucleic acid has the nucleic acid sequence of SEQ ID NO: 5 where at least one codon encoding one of amino acids 681 -684 of SEQ ID NO:6 is mutated such the encoded MEKKl protein is resistant to proteolysis by a caspase after an amino acid equivalent to amino acid 684 of SEQ ID NO:6.
  • the invention provides a method of stimulating apoptosis in a cell comprising introducing into the cell an expression vector encoding an MEKKl active fragment of the invention such that MEKKl active fragment is produced in the cell and apoptosis is stimulated.
  • the invention provides a method of inhibiting apoptosis in a cell comprising introducing into the cell an expression vector encoding a protease-resistant MEKKl protein of the invention such that protease- resistant MEKKl protein is produced in the cell and apoptosis is inhibited.
  • an MEKKl active fragment can be generated in vitro by: - 9 -
  • the caspase protease is a caspase-3 protease.
  • the caspase protease is a caspase-7 protease. Standard proteolysis conditions known in the art under which caspase proteases are known to be active can be used in the method of the invention.
  • the invention provides a method of identifying a compound that modulates the apoptotic activity of an MEKKl active fragment.
  • the method comprises: providing an indicator cell that comprises an MEKKl active fragment of the invention; contacting the indicator cell with a test compound; and determining the effect of the test compound on the apoptotic activity of the MEKKl active fragment in the indicator cell to thereby identify a compound that modulates the apoptotic activity of the MEKKl active fragment.
  • the indicator cell may naturally express an MEKKl active fragment or may be transfected with an expression vector that encodes the MEKKl active fragment such that the active fragment is expressed in the cell.
  • the effect of the test compound can be evaluated, for example, by measuring an apoptotic response in the cells, such as DNA fragmentation.
  • the invention provides a method of identifying a compound that modulates the proteolytic cleavage of an MEKKl protein by a caspase protease, comprising: providing a reaction mixture that comprises an MEKKl protein and a caspase protease; contacting the reaction mixture with a test compound; and determining the effect of the test compound on proteolytic cleavage of the
  • MEKKl protein by the caspase protease to thereby identify a compound that modulates the proteolytic cleavage of an MEKKl protein by a caspase protease.
  • the caspase protease is a caspase-3 protease.
  • the caspase protease is a caspase-7 protease.
  • Standard proteolysis conditions known in the art under which caspase proteases are known to be active can be used in the method of the invention.
  • the effect of the test compound on the proteolytic cleavage of MEKKl can be evaluated by, for example, monitoring the generation of the 91 kD active - 10 -
  • Figure 1 depicts the cDNA sequence of human MEKKl.
  • the nucleotide sequence corresponds to nucleic acids 1 to 3911 of SEQ ID NO:5.
  • Figure 2 depicts the cDNA sequence of murine MEKKl.
  • the nucleotide sequence corresponds to nucleic acids 1 to 5253 of SEQ ID NO:3.
  • Figure 3 depicts an alignment of the amino acid sequences of murine MEKKl (amino acids 1-1493 of SEQ ID NO:4 and human MEKKl (amino acids 1-1303 of SEQ ID NO: 6). The conserved caspase cleavage site is boxed. Amino acids which are unique as between murine and human MEKKl are underlined.
  • Figure 4 is a schematic representation of the HA-tagged mouse MEKKl protein showing the regions (the numbers correspond to the position of the amino acids) used to generate the indicated antibodies. Also shown is the sequence (one letter code) between amino acids 853 and 888 of SEQ ID NO:4 where the tetrapeptides DEVE (SEQ ID NO: 7) and DTVD (SEQ ID NO: 8) (in bold) have been replaced with alanine residues in mutants DEVE ⁇ A and DTVD ⁇ A, respectively.
  • Figure 5 is a schematic representation of the p35-inhibitable and p35 -insensitive cleavage in the mouse MEKKl protein.
  • the letters A to D indicate the names of the cleavage products.
  • the molecular weights were calculated from the migration of the markers in at least 2 different experiments.
  • Figure 6 is a schematic diagram of a mechanistic model of MEKKl -induced apoptosis.
  • Figure 7 depicts an alignment of the amino acid sequences of murine MEKKl and rat MEKKl (having Accession No. Q62925).
  • the rat MEKKl amino acid sequence is set forth as SEQ ID NO:21.
  • the predicted caspase cleavage site in rat is boxed.
  • a predicted rat apoptotic fragment begins after the cleavage site and comprises amino acid residues 875-1493 of SEQ ID NO-21.
  • Figure 8 depicts an alignment of the amino acid sequences of murine MEKKl, rat MEKKl and partial amino acid sequences of human MEKKl.
  • MEKK kinases novel mitogen ERK kinase kinase proteins
  • MEKKs mitogen ERK kinase kinase proteins
  • certain aspects of the present invention relate to nucleic acids encoding vertebrate MEKKl proteins (e.g., human and murine MEKKl proteins), the MEKKl proteins themselves, antibodies immunoreactive with MEKKl proteins, and preparations of such compositions.
  • the present invention provides diagnostic and therapeutic assays and reagents for detecting and treating disorders involving, for example, aberrant expression or activation of the MEKKl gene products.
  • drug discovery assays are provided for identifying agents which can modulate the biological function of MEKKl proteins, such as by altering the binding of the protein to either downstream or upstream elements in a signal transduction pathway, or which inhibit the kinase activity of the MEKKl protein. Such agents can be useful therapeutically to alter the growth and/or differentiation of a cell.
  • Other aspects of the invention are described below or will be apparent to those skilled in the art in light of the present disclosure.
  • isolated MEKK proteins As used herein protein, peptide, and polypeptide are meant to be synonymous. According to the present invention, an isolated protein is a protein that has been removed from its natural milieu. It will be understood that "isolated”, with respect to MEKK polypeptides, is - 12 -
  • isolated MEKK protein preparations include preparations having less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • Functional forms of the subject MEKK polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
  • the subject MEKK polypeptides can be isolated by affinity purification using, for example, a catalytically inactive MEK.
  • Isolated does not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g., lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g., acrylamide or agarose) substances or solutions.
  • an isolated MEKK protein can, for example, be obtained from its natural source, be produced using recombinant DNA technology, or be synthesized chemically.
  • an isolated MEKK protein can be a full-length MEKK protein or any homologue of such a protein, such as a MEKK protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycosylphosphatidyl inositol), wherein the modified protein retains a MEKK biological activity (e.g., is capable of phosphorylating MAP kinase kinases, such as mitogen ERK kinases (MEKs (MKKl and MKK2)) and/or Jun kinase
  • a homologue of a MEKK protein is a protein having an amino acid sequence that is substantially similar or homologous to a natural MEKK protein amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e. , with) a nucleic acid sequence encoding the natural MEKK protein amino acid sequence.
  • stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et ⁇ l. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989).
  • Exemplary stringent hybridization conditions include but are not limited to hybridization at 65°C in 4X SSC or at 42°C in 4XSSC, 50% formamide, followed by washing at 65°C in lXSSC. - 13 -
  • Exemplary high stringency conditions include but are not limited to hybridization at 65° C in 1XSSC or at 42°C in 1XSSC, 50% formamide followed by washing at 65°C in 0.3XsSSC.
  • a homologue of a MEKK protein also includes a protein having an amino acid sequence that is sufficiently cross-reactive such that the homologue has the ability to elicit an immune response against at least one epitope of a naturally-occurring MEKK protein.
  • the minimal size of a protein homologue of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein.
  • the size of the nucleic acid molecule encoding such a protein homologue is dependent on nucleic acid composition, percent homology between the nucleic acid molecule and complementary sequence, as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration).
  • the minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich.
  • the minimal size of a nucleic acid molecule used to encode a MEKK protein homologue of the present invention is from about 12 to about 18 nucleotides in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof.
  • the minimal size of a MEKK protein homologue of the present invention is from about 4 to about 6 amino acids in length, with preferred sizes depending on whether a full-length, multivalent protein (i.e., fusion protein having more than one domain each of which has a function), or a functional portion of such a protein is desired.
  • a homologue of a MEKK protein is a protein having an amino acid sequence that is at least about 60-65%, 70-75%, 80-85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6 or a portion or fragment thereof.
  • a MEKK homologue is a protein which is encoded by a nucleic acid molecule having at least 60-65%, 70-75%, 80-85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:5.
  • % homology can be used interchangeably with the term "% identity”.
  • the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%>, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90%> of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W.
  • nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • BLAST nucleotide searches can be performed with the - 15 -
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g. , XBLAST and NBLAST can be used. See http://www.ncbi.nlm.nih.gov.
  • MEKK protein homologues can be the result of allelic variation of a natural gene encoding a MEKK protein.
  • a natural gene refers to the form of the gene found most often in nature.
  • MEKK protein homologues can be produced using techniques known in the art including, but not limited to, direct modifications to a gene encoding a protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • mutagenesis includes point mutations, as well as deletions and truncations of the MEKK polypeptide sequence.
  • the ability of a MEKK protein homologue to phosphorylate MEK and/or JNKK protein can be tested using techniques known to those skilled in the art.
  • the structure of the subject MEKK polypeptides for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
  • modified polypeptides when designed to retain at least one activity of the naturally-occurring form of the protein, are considered functional equivalents of the MEKK polypeptide described in more detail herein.
  • modified peptide can be produced, for instance, by amino acid substitution, deletion, or addition.
  • the MEKK proteins and/or MEKK homologues are defined as having a MEKK "activity" or "biological activity”.
  • the MEKK protein is involved in a pathway controlling the phosphorylation of a mitogen-activated protein (MAP) kinase.
  • MAP mitogen-activated protein
  • the mammalian MAP kinase family includes, for example, the extracellular signal -regulated protein kinases (ERK1 and ERK2), p42 or p44 MAPKs.
  • the MEKK protein will be involved in the pathway controlling c-Jun NH2-terminal kinases (JNKs, or SAPKs), and the so- called "p38 subgroup" kinases (p38 and Hog-1 kinases).
  • MEKK proteins of the present invention interact with, and directly phosphorylate members of the MAP kinase kinase family (MEKs or MKKs), as MEK1, MEK2, MKKl , MKK2, or the stress-activated kinases (SEKs), and the Jun kinase kinases (JNKK1, JNKK2, MKK3, MKK4), or the like.
  • MEKs or MKKs MAP kinase kinase family
  • SEKs stress-activated kinases
  • JNKK1, JNKK2, MKK3, MKK4 Jun kinase kinases
  • a MEKK protein is capable of regulating the activity of signal transduction proteins including, but not limited to, mitogen activated ERK kinases (MEKs), mitogen activated protein kinases (MAPKs), transcription control factor (TCF), Ets-like-1 transcription factor (Elk-1), Jun ERK kinases (JNKKs), Jun kinases (JNK; which is equivalent to SAPK), stress activated MAPK proteins, Jun, activating transcription factor-2 (ATF-2) and/or Myc protein.
  • MEKs mitogen activated ERK kinases
  • MAPKs mitogen activated protein kinases
  • MAPKs mitogen activated protein kinases
  • TCF transcription control factor
  • Elk-1 Ets-like-1 transcription factor
  • JNKKs Jun ERK kinases
  • JNK Jun kinases
  • the "activity" or “biological activity” of a protein can be directly correlated with the phosphorylation state of the protein and/or the ability of the protein to perform a particular function (e.g., phosphorylate another protein or regulate transcription).
  • Preferred MEK proteins regulated by a MEKK protein of the present invention include MEK-1 and/or MEK-2 (MKKl or MKK2).
  • Preferred MAPK proteins regulated by a MEKK protein of the present invention include p38/Hog-l MAPK, p42 MAPK and/or p44 MAPK.
  • Preferred stress activated MAPK proteins regulated by a MEKK protein of the present invention include Jun kinase (JNK), stress activated MAPK- ⁇ and/or stress activated MAPK- ⁇ .
  • a MEKK protein of the present invention is capable of phosphorylating a MEK or MKK, Jun kinase kinase (JNKK) and/ or stress activated ERK kinase (SEK), in particular MEK1, MEK2, MKKl, MKK2, MKK3, MKK4, JNKKl, JNKK2, SEK1 and/or SEK2 proteins.
  • JNKK Jun kinase kinase
  • SEK stress activated ERK kinase
  • MEK1 and MEK2 are equivalent to MKKl and MKK2, respectively.
  • JNKKl and JNKK2 are equivalent to MKK3 and MKK4, which are equivalent to SEK1 and SEK2.
  • a preferred MEKK protein of the present invention is additionally capable of inducing Myc proteins and members of the Ets family of transcription factors, such as TCF protein, also referred to as Elk-1 protein.
  • Another aspect of the present invention is the recognition that a MEKK protein of the present invention is capable of regulating the apoptosis of a cell
  • apoptosis refers to the form of cell death that comprises: progressive contraction of cell volume with the preservation of the integrity of cytoplasmic organelles; condensation of chromatin, as viewed by light or electron microscopy; and DNA cleavage, as electrophoresis or labeling of DNA fragments using terminal deoxytransferase (TDT).
  • TDT terminal deoxytransferase
  • a preferred MEKK protein of the present invention is capable of inducing the apoptosis of cells, such that the cells have characteristics substantially similar to cytoplasmic shrinkage and/or nuclear condensation. - 17 -
  • FIG. 6 A schematic representation of exemplary cell growth regulatory pathways that are MEKK dependent is shown in Figure 6.
  • a MEKK protein of the present invention comprises numerous unique structural characteristics.
  • a MEKK protein of the present invention includes at least one of two different structural domains having particular functional characteristics.
  • Such structural domains include an NH2-terminal regulatory domain that serves to regulate a second structural domain comprising a COOH-terminal protein kinase catalytic domain that is capable of phosphorylating an MKK protein.
  • a MEKK protein of the present invention includes a full-length MEKK protein, as well as at least a portion of a MEKK protein capable of performing at least one of the functions defined above. Preferred portions are capable of inducing apoptosis.
  • a MEKK protein refers to a portion of a MEKK protein encoded by a nucleic acid molecule that is capable of hybridizing, under stringent conditions, with a nucleic acid encoding a full-length MEKK protein of the present invention. Preferred portions of MEKK proteins are useful for regulating apoptosis in a cell.
  • Suitable sizes for portions of a MEKK protein of the present invention are 65-70kD, 75-80kD, 85-90kD, 100-1 lOkD, 120-130kD, 140- 150kD, 160-170kD or 180kD or larger as determined by Tris-glycine SDS-PAGE, preferably using an 8% polyacrylamide SDS gel (SDS-PAGE) and resolved using methods standard in the art. It is noted that experimental conditions used when running gels to determine the molecular size of putative MEKK proteins and/or portions thereof will cause variations in results.
  • a portion of a MEKK protein capable of inducing apoptosis includes about amino acids 875-1493 of murine MEKKl, set forth in SEQ ID NO:4. In another embodiment, a portion of a MEKKl protein capable of inducing apoptosis includes about amino acids 685-1303 of human MEKKl, set forth in SEQ ID NO:6. In another embodiment, a portion of a MEKK protein capable of inducing apoptosis is substantially similar or homologous to amino acids 875-1493 of murine MEKKl, set forth in SEQ ID NO:4. In another embodiment, a portion of a MEKK protein capable of inducing apoptosis is substantially similar or homologous to amino acids 685-1303 of human MEKKl, set forth in SEQ ID NO:6.
  • the sequences comprising the catalytic domain of a MEKK protein are involved in phosphotransferase activity, and therefore display a relatively conserved amino acid sequence.
  • the NH2-terminal regulatory domain of a MEKK protein can be substantially divergent. As such, the NH 2 -terminal regulatory domain of a MEKK - 18 -
  • the subject MEKK proteins are provided as fusion proteins.
  • fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the MEKK polypeptides of the present invention.
  • MEKK polypeptides can be generated as glutathione-S-transferase (GST-fusion) proteins.
  • GST-fusion proteins can enable easy purification of the MEKK polypeptide, as for example by the use of glutathione- derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified protein (e.g., see Hochuli et al. (1987) J. Chromatography 411 :177; and Janknecht et al. PNAS 88:8972).
  • fusion genes are known to those skilled in the -art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt- ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • a MEKK protein of the present invention can include MEKK proteins that have undergone post-translational modification. Such modification can include, for example, phosphorylation or among other post- translational modifications including conformational changes or post-translational deletions.
  • This invention further contemplates a method for generating sets of combinatorial mutants of the subject MEKK proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g., homologs) that are - 19 -
  • MEKK homologs can act as either agonists or antagonist of the wild-type MEKK proteins, or alternatively, which possess novel activities all together.
  • MEKK homologs can be engineered by the present method to provide selective, constitutive activation of a pathway, so as mimic induction by a factor when the MEKK homolog is expressed in a cell capable of responding to the factor.
  • combinatorially-derived homologs can be generated to have an increased potency relative to a naturally occurring form of the protein.
  • MEKK homologs can be generated by the present combinatorial approach to selectively inhibit (antagonize) induction by a growth or other factor.
  • mutagenesis can provide MEKK homologs which are able to bind other signal pathway proteins (e.g., MEKs) yet prevent propagation of the signal, e.g., the homologs can be dominant negative mutants.
  • manipulation of certain domains of MEKK by the present method can provide domains more suitable for use in fusion proteins.
  • the amino acid sequences for a population of MEKK homologs or other related proteins are aligned, preferably to promote the highest homology possible.
  • a population of variants can include, for example, MEKK homologs from one or more species.
  • Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.
  • the variegated library of MEKK variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential MEKK sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of MEKK sequences therein.
  • a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of a MEKK coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with SI nuclease; and (v) ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of MEKK homologs.
  • the most widely used techniques for screening large gene libraries typically comprise cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • the gene library can be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • E coli filamentous phages Ml 3, fd, and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02909; Garrard et al, PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson et al (1991) Nature 352:624-628; and Barbas et al.
  • the resulting phage libraries with the fusion tail proteins may be panned, e.g., using a fluorescent labeled M ⁇ K protein, e.g., FITC-M ⁇ K, to score for M ⁇ KK homologs which retain the ability to bind to the M ⁇ K protein.
  • a fluorescent labeled M ⁇ K protein e.g., FITC-M ⁇ K
  • Individual phage which encode a M ⁇ KK homolog which retains M ⁇ K binding can be isolated, the M ⁇ KK homolog gene recovered from the isolate, and further tested to discern between active and antagonistic mutants
  • cells e.g., R ⁇ F52 cells
  • the library of expression vectors can be transfected into a population of REF52 cells which also inducibly overexpress a MEKK protein (e.g., and which overexpression causes apoptosis). Expression of WT-MEKK is then induced, and the effect of the MEKK mutant on induction of apoptosis can be detected. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of apoptosis, and the individual clones further characterized.
  • the invention also provides for reduction of the MEKK proteins to generate mimetics, e.g., peptide or non-peptide agents, which are able to disrupt binding of a MEKK polypeptide of the present invention with either upstream or downstream components of its signaling cascade.
  • mimetics e.g., peptide or non-peptide agents
  • Such mutagenic techniques as described above are also useful to map the determinants of the MEKK proteins which participate in protein-protein interactions involved in, for example, binding of the subject MEKK polypeptide to proteins which may function upstream (including both activators and repressors of its activity) or to proteins which may function downstream of the MEKK polypeptide, whether they are positively or negatively regulated by it.
  • the critical residues of a subject MEKK polypeptide which are involved in molecular recognition of an upstream or downstream MEKK component can be determined and used to generate MEKK-derived peptidomimetics which competitively inhibit binding of the authentic protein with that moiety.
  • peptidomimetic compounds can be generated which mimic those residues of the MEKK protein which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of a - 22 -
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R.
  • an isolated nucleic acid molecule capable of hybridizing, under stringent conditions, with a MEKK protein gene encoding a MEKK protein of the present invention.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation).
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • isolated refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
  • an isolated nucleic acid encoding one of the subject MEKK polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the MEKK gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
  • the isolated MEKK nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • isolated will also be understood to include nucleic acid that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.
  • nucleic acid includes polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • the term “gene” or “recombinant gene” includes nucleic acid comprising an open reading frame encoding one of the MEKK polypeptides of the present invention, including both exon and (optionally) intron sequences.
  • recombinant gene refers to nucleic acid encoding a MEKK polypeptide (e.g., a vertebrate MEKK polypeptide) and comprising MEKK-encoding exon sequences, though it may optionally include intron sequences which are either derived from a chromosomal MEKK gene or from an unrelated chromosomal gene.
  • exemplary recombinant genes encoding the subject MEKK polypeptides are represented in the appended Sequence Listing.
  • the term "intron” refers to a DNA sequence present in a given MEKK gene which is not translated into protein and is generally found between exons.
  • An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene.
  • the phrase "at least a portion of an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity.
  • at least a portion of a nucleic acid sequence is an amount of a nucleic acid sequence capable of forming a stable hybrid with a particular desired gene (e.g. , MEKK genes) under stringent hybridization conditions.
  • Isolated MEKK protein nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a MEKK protein of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates of MEKK.
  • Preferred modifications to a MEKK protein nucleic acid molecule of the present invention include truncating a full-length MEKK protein nucleic acid molecule by, for example: deleting at least a portion of a MEKK protein nucleic acid molecule encoding - 24 -
  • a regulatory domain to produce a constitutively active MEKK protein deleting at least a portion of a MEKK protein nucleic acid molecule encoding a catalytic domain to produce an inactive MEKK protein; and modifying the MEKK protein to achieve desired inactivation and/or stimulation of the protein, for example, substituting a codon encoding a lysine residue in the catalytic domain (/. e. , phosphotransferase domain) with a methionine residue to inactivate the catalytic domain.
  • a preferred truncated MEKK nucleic acid molecule encodes a form of a MEKK protein which is capable of inducing apoptosis.
  • An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one MEKK protein of the present invention, examples of such proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides that comprise the nucleic acid molecule, the two phrases can be used interchangeably.
  • MEKK proteins of the present invention include, but are not limited to, proteins having full-length MEKK protein coding regions, portions thereof, and other MEKK protein homologues.
  • a MEKK protein gene includes all nucleic acid sequences related to a natural MEKK protein gene such as regulatory regions that control production of a MEKK protein encoded by that gene (including, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • a nucleic acid molecule of the present invention can be an isolated natural MEKK protein nucleic acid molecule or a homologue thereof.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of a MEKK protein nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a corresponding natural gene.
  • a MEKK protein nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, e.g., Sambrook et al, ibid.).
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • Nucleic acid molecule homologues can be selected from a - 25 -
  • a preferred nucleic acid molecule of the present invention is capable of hybridizing under stringent conditions to a nucleic acid that encodes at least a portion of a MEKK protein, or a homologue thereof. Also preferred is a MEKK nucleic acid molecule that includes a nucleic acid sequence having at least about 50% homology, preferably 75% homology, preferably 85% homology, or even more preferably 95% homology with an MEKK nucleic acid molecule of the invention.
  • nucleic acids have 50%, preferably at least about 75%, and more preferably at least about 85%, and most preferably at least about 95% homology with the corresponding region(s) of the nucleic acid sequence encoding the catalytic domain of a MEKK protein, or a homologue thereof.
  • a MEKK protein nucleic acid molecule that includes a nucleic acid sequence having at least about 50%, preferably at least about 15%, more preferably at least about 85%, and even more preferably at least about 95% homology with the corresponding region(s) of the nucleic acid sequence encoding the NH2-terminal regulatory domain of a MEKK protein, or a homologue thereof.
  • Such nucleic acid molecules can be a full-length gene and/or a nucleic acid molecule encoding a full-length protein, a hybrid protein, a fusion protein, a multivalent protein or a truncation fragment.
  • nucleic acid molecule of a MEKK protein of the present invention allows one skilled in the art to make copies of that nucleic acid molecule as well as to obtain additional portions of MEKK protein-encoding genes (e.g., nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or MEKK protein nucleic acid molecule homologues. Knowing a portion of an amino acid sequence of a MEKK protein of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such a MEKK protein.
  • the present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of a MEKK protein, or a homologue thereof.
  • a preferred oligonucleotide is capable of hybridizing, under stringent conditions, with a nucleic acid molecule of SEQ ID NO: 3 or SEQ ID NO:5.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
  • the minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another - 26 -
  • nucleic acid molecule of the present invention nucleic acid molecule of the present invention.
  • Minimal size characteristics of preferred oligonucleotides are at least about 10 nucleotides, preferably at least about 20 nucleotides, more preferably at least about 50 nucleotides and most preferably at least about 60 nucleotides. Larger fragments are also contemplated.
  • the size of the oligonucleotide must also be sufficient for the use of the oligonucleotide in accordance with the present invention.
  • Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit, for example, expression of MEKK proteins by cells.
  • Such therapeutic applications include the use of such oligonucleotides in, for example, antisense-, triplex formation-, ribozyme- and/or RNA drug-based technologies.
  • the present invention therefore, includes use of such oligonucleotides and methods to interfere with the production of MEKK proteins.
  • oligonucleotides encoding portions of MEKK proteins which bind to MEKK binding proteins can be used a therapeutics.
  • the peptides encoded by these nucleic acids are used.
  • antisense therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g., bind) under cellular conditions, with the cellular mRNA -and/or genomic DNA encoding one or more of the subject MEKK proteins so as to inhibit expression of that protein, e.g., by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
  • An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a vertebrate MEKK protein.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a vertebrate MEKK gene.
  • oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo.
  • exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and - 27 -
  • the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, the oligomers of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration.
  • the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.
  • the antisense constructs of the present invention by antagonizing the normal biological activity of one of the MEKK proteins, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures.
  • tissue e.g., tissue differentiation
  • the anti-sense techniques e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a MEKK mRNA or gene sequence
  • the present invention also includes a recombinant vector which includes at least one MEKK protein nucleic acid molecule of the present invention inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, for example nucleic acid sequences that are not naturally found adjacent to MEKK protein nucleic acid molecules of the present invention.
  • the vector can be either RNA or DNA, and either prokaryotic or eukaryotic, and is typically a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of MEKK protein nucleic acid molecules of the present invention.
  • recombinant vector herein referred to as a recombinant molecule and described in more detail below, can be used in the expression of nucleic acid molecules of the present invention.
  • Preferred recombinant vectors are capable of replicating in the transformed cell.
  • Preferred nucleic acid molecules to insert into a recombinant vector includes a nucleic acid molecule that encodes at least a portion of a MEKK protein, or a homologue thereof.
  • portions of a MEKK nucleic acid which encodes a MEKK catalytic domain is used.
  • at least a portion of a nucleic acid which encodes the portion of a MEKK protein which binds to a MEKK substrate or a MEKK regulatory protein is used.
  • Preferred nucleic acid molecules for insertion into an expression vector include nucleic acid molecules that encode at least a portion of a MEKK protein, or a homologue thereof.
  • Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins.
  • Inclusion of a fusion sequence as part of a MEKK nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protein encoded by the nucleic acid molecule.
  • a fusion segment can function as a tool to simplify purification of a MEKK protein, such as to enable purification of the resultant fusion protein using affinity chromatography.
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g. , increased stability and/or purification tool). It is within the scope of the present invention to use one or more fusion segments.
  • Fusion segments can be joined to amino and/or carboxyl termini of a MEKK protein.
  • Linkages between fusion segments and MEKK proteins can be constructed to be susceptible to cleavage to enable straight- forward recovery of the MEKK proteins.
  • Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that - 29 -
  • the gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of one of the subject MEKK proteins.
  • another aspect of the invention features expression vectors for in vivo or in vitro transfection and expression of a MEKK polypeptide in particular cell types so as to reconstitute the function of, constitutively activate, or alternatively, abrogate the function of a signal pathway dependent on a MEKK activity.
  • Such therapies may useful where the naturally- occurring form of the protein is misexpressed or inappropriately activated; or to deliver a form of the protein which alters differentiation of tissue; or which inhibits neoplastic transformation.
  • Expression constructs of the subject MEKK polypeptide, and mutants thereof, may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively delivering the recombinant gene to cells in vivo.
  • Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPU4 precipitation carried out in vivo.
  • transduction of appropriate target cells represents the critical first step in gene therapy
  • choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g., locally or systemically.
  • the particular gene construct provided for in vivo tr-ansduction of MEKK expression are also useful for in vitro transduction of cells, such as for use in the ex vivo tissue culture systems described below.
  • a preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA, encoding the particular MEKK polypeptide desired.
  • Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid. - 30 -
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the subject proteins rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
  • Retroviruses have been used to introduce a variety of genes into many different cell types, including neuronal cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al (1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983) Virology 163 :251 -254); or coupling cell surface receptor ligands to the viral env proteins (Neda et al. (1991) J Biol Chem 266:14143-14146).
  • Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g., single- chain antibody/e/w fusion proteins).
  • a protein or other variety e.g., lactose to convert the env protein to an asialoglycoprotein
  • fusion proteins e.g., single- chain antibody/e/w fusion proteins
  • retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the MEKK gene of the retroviral vector.
  • Another viral gene delivery system useful in the present invention utilizes adeno virus-derived vectors.
  • the genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) Biotechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl Acad. Sci.
  • virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA and foreign DNA contained therein is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El - 32 -
  • MEKK major late promoter
  • E3 E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et al, supra; and Graham et al. in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127).
  • Expression of the inserted MEKK gene can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
  • MLP major late promoter
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • Adeno-associated virus is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.
  • AMINO ACIDSV vector such as that described in Tratschin et al (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AMINO ACIDSV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.
  • non-viral methods can also be employed to cause expression of a subject MEKK polypeptide in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject MEKK polypeptide gene by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
  • the gene delivery systems for the therapeutic MEKK gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S.
  • a MEKK gene such as any one of the clones represented in the appended Sequence Listing, can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • Still another aspect of the present invention pertains to recombinant cells, e.g. , cells which are transformed with at least one of any nucleic acid molecule of the present invention.
  • a preferred recombinant cell is a cell transformed with at least one nucleic acid molecule that encodes at least a portion of a MEKK protein, or a homologue thereof.
  • Suitable host cells for transforming a cell can include any cell capable of producing MEKK proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule.
  • Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells.
  • Preferred host cells include bacterial, yeast, insect and mammalian cells, with mammalian cells being particularly preferred.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, insect, animal, and/or plant cells.
  • nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention.
  • transcription control sequences include those which function in bacterial, yeast, and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda ( ⁇ ) (such as ⁇ p L and ⁇ p R and fusions that include such promoters), bacteriophage T7, ⁇ llac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha mating factor, baculovirus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences, as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding a MEKK protein.
  • Recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post- translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control - 35 -
  • nucleic acid molecules of the present invention may correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant protein production during fermentation.
  • the activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing the resultant protein.
  • amplifying the copy number of a nucleic acid sequence in a cell can be accomplished either by increasing the copy number of the nucleic acid sequence in the cell's genome or by introducing additional copies of the nucleic acid sequence into the cell by transformation.
  • Copy number amplification is conducted in a manner such that greater amounts of enzyme are produced, leading to enhanced conversion of substrate to product.
  • recombinant molecules containing nucleic acids of the present invention can be transformed into cells to enhance enzyme synthesis.
  • Transformation can be accomplished using any process by which nucleic acid sequences are inserted into a cell.
  • the nucleic acid sequence on the recombinant molecule can be manipulated to encode an enzyme having a higher specific activity.
  • recombinant cells can be used to produce a MEKK protein of the present invention by culturing such cells under conditions effective to produce such a protein, and recovering the protein.
  • Effective conditions to produce a protein include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing a MEKK protein.
  • Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • the medium may comprise complex nutrients or may be a defined minimal medium.
  • a preferred cell to culture is a recombinant cell that is capable of expressing the MEKK protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, micro injection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, - 36 -
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur.
  • the cells may be harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • the recombinant MEKK polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the -art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.
  • the recombinant MEKK polypeptide is a fusion protein containing a domain which facilitates its purification, such as GST fusion protein or poly(His) fusion protein.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.
  • resultant MEKK proteins may either remain within the recombinant cell or be secreted into the fermentation medium.
  • the phrase "recovering the protein” refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification.
  • MEKK proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing and differential solubilization.
  • a MEKK protein of the present invention can be produced by isolating the MEKK protein from cells or tissues recovered from an animal that normally express the MEKK protein.
  • a cell type such as T cells
  • MEKK protein can then be isolated from the isolated primary T cells using standard techniques described herein. - 37 -
  • MEKK polypeptides as described in the present invention facilitates the development of assays which can be used to screen for drugs, including MEKK homologs, which are either agonists or antagonists of the normal cellular function of the subject MEKK polypeptides, or of their role in the pathogenesis of cellular differentiation and/or proliferation, and disorders related thereto.
  • the assay evaluates the ability of a compound to modulate binding between a MEKK polypeptide and a molecule that interacts either upstream or downstream of the MEKK polypeptide in the a cellular signaling pathway.
  • assay formats will suffice and, in light of the present inventions, will be comprehended by a skilled artisan.
  • the compound of interest is contacted with proteins which may function upstream (including both activators and repressors of its activity such as, Ras, Rac, Cdc 42 or Rho or other Ras superfamily members) or to proteins or nucleic acids which may function downstream of the MEKK polypeptide, whether they are positively or negatively regulated by it.
  • proteins which may function upstream including both activators and repressors of its activity such as, Ras, Rac, Cdc 42 or Rho or other Ras superfamily members
  • proteins or nucleic acids which may function downstream of the MEKK polypeptide, whether they are positively or negatively regulated by it.
  • MEKK-bp such polypeptides of a signal transduction pathway which interact directly with MEKK
  • MEKKs members of the MAP kinase kinase family (MEKs or MKKs), as MEK1, MEK2, MKKl , MKK2, the stress-activated kinases (SEKs), also known as the Jun kinase kinases (JNKKs), MEKK3 and MEKK4 or the like.
  • MEKKs members of the MAP kinase kinase family
  • SEKs stress-activated kinases
  • JNKKs Jun kinase kinases
  • MEKK family proteins from the mammalian MAP kinase family which includes, for example, the extracellular signal-regulated protein kinases (ERKs), c-Jun NH 2 -terminal kinases (JNKs, or SAPKs), and the so-called "p38 subgroup” kinases (p38 kinases).
  • ERKs extracellular signal-regulated protein kinases
  • JNKs c-Jun NH 2 -terminal kinases
  • SAPKs c-Jun NH 2 -terminal kinases
  • p38 subgroup kinases
  • a composition containing a MEKK polypeptide is then added to the mixture of the compound and the MEKK-bp.
  • Detection and quantification of complexes including MEKK and the MEKK-bp provide a means for determining a compound's efficacy at inhibiting (or potentiating) complex formation between MEKK and the MEKK-binding protein.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified MEKK polypeptide is added to a composition containing the MEKK-binding protein, and the formation of a complex is quantitated in the absence of the test compound.
  • Complex formation between the MEKK polypeptide and a MEKK-binding protein may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled MEKK polypeptides, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled MEKK polypeptides
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/MEKK GST/MEKK
  • GST/MEKK glutathione-S-transferase/MEKK
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g., beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of MEKK-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
  • Other techniques for immobilizing proteins on matrices are also available for use in the subject assay.
  • either MEKK or its cognate binding protein can be immobilized utilizing conjugation of biotin and streptavidin.
  • MEKK molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • antibodies reactive with MEKK but which do not interfere with binding of upstream or downstream elements can be derivatized to the wells of the plate, and MEKK trapped in the wells by antibody conjugation.
  • preparations of a MEKK-binding protein and a test compound are incubated in the MEKK-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the MEKK binding protein, or which are reactive with the MEKK protein and compete with the binding protein; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding protein, either intrinsic or extrinsic activity.
  • the enzyme can be chemically conjugated or provided as a fusion protein with the MEKK-bp.
  • the MEKK-bp can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g., 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-napthol.
  • a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
  • antibodies against the protein can be used.
  • the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the MEKK sequence, a second polypeptide for which antibodies are readily available (e.g., from commercial sources).
  • the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
  • myc-epitopes e.g., see Ellison et al (1991) J Biol Chem 266:21150-21157
  • pFLAG system International Biotechnologies, Inc.
  • pEZZ-protein A system Pharamacia, NJ
  • the readily available source of vertebrate MEKK proteins provided by the present invention also facilitates the generation of cell-based assays for identifying small molecule agonists/antagonists and the like.
  • MEKK-dependent signal transduction can be caused to overexpress a recombinant MEKK protein in the presence and absence of a test agent of interest, with the assay scoring for modulation in MEKK-dependent responses by the target cell mediated by the test agent.
  • agents which produce a statistically significant change in MEKK-dependent signal transduction can be identified.
  • a two hybrid assay can be generated with a MEKK and MEKK-binding protein.
  • This assay permits the detection of protein- protein interactions in yeast such that drug dependent inhibition or potentiation of the interaction can be scored.
  • GAL4 protein is a potent activator of transcription in yeast grown on galactose. The ability of GAL4 to activate transcription depends on the presence of an N-terminal sequence capable of binding to a specific DNA sequence (UASG) and a C-teminal domain containing a transcriptional activator.
  • a sequence encoding a MEKK protein, "A” may be fused to that encoding the DNA binding domain of the GAL4 protein.
  • a second hybrid protein may be created by fusing sequence encoding the GAL4 transactivation domain to sequence encoding a MEKK-bp, "B". If protein "A” and protein "B" interact, that interaction serves to bring together the two domains of GAL4 necessary to activate transcription of a UASG- containing gene.
  • yeast strains appropriate for the detection of protein-protein interactions would contain, for example, a GALl-lacZ fusion gene to permit detection of transcription from a UASG sequence.
  • Other examples of two-hybrid assays or interaction trap assays are known in the art.
  • a portion of MEKK4 providing a Rac/Cdc42 binding site is provided in one fusion protein, along with a second fusion protein including a Rac/Cdc42 polypeptide.
  • This embodiment of the subject assay permits the screening of compounds which inhibit or potentiate the binding of MEKK4 and Cdc42. Phosphorylation assays may also be used.
  • MEKK binding proteins can be tested for their ability to phosphorylate substrates in addition, compounds that inhibit or activate MEKK regulated pathways and phenotypic responses can be tested.
  • each of the assay systems set out above can be generated in a
  • the assay format can provide information regarding specificity as well as potency. For instance, side-by-side comparison of a test compound's effect on different MEKKs can provide information on selectivity, and permit the identification of compounds which selectively modulate the bioactivity of only a subset of the MEKK family. - 41 -
  • the present invention also includes a method to identify compounds capable of regulating signals initiated from a receptor on the surface of a cell, such signal regulation involving in some respect, MEKK protein.
  • a method comprises the steps of: (a) contacting a cell containing a MEKK protein with a putative regulatory compound; (b) contacting the cell with a ligand capable of binding to a receptor on the surface of the cell; and (c) assessing the ability of the putative regulatory compound to regulate cellular signals by determining activation of a member of a MEKK-dependent pathway of the present invention.
  • a preferred method to perform step (c) comprises measuring the phosphorylation of a member of a MEKK-dependent pathway.
  • step (c) comprises measuring the ability of the MEKK protein to phosphorylate a substrate molecule comprising a protein including MKKl, MKK2, MKK3, or MKK4, Raf-1, Ras- GAP and neurofibromin using methods described herein.
  • Preferred substrates include MEKl , MEK2, JNKKl and JNKK2.
  • step (c) comprises determining the ability of MEKK protein to bind to Ras, rac or Cdc 42 protein. In particular, determining the ability of MEKK protein to bind to GST-Ras vl2 (GTP ⁇ S) or GST-Rac vl (GTP ⁇ S).
  • Putative compounds as referred to herein include, for example, compounds that are products of rational drug design, natural products and compounds having partially defined signal transduction regulatory properties.
  • a putative compound can be a protein-based compound, a carbohydrate-based compound, a lipid-based compound, a nucleic acid-based compound, a natural organic compound, a synthetically derived organic compound, an anti-idiotypic antibody and/or catalytic antibody, or fragments thereof.
  • a putative regulatory compound can be obtained, for example, from libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (/. e. , libraries of compounds that differ in sequence or size but that have the same building blocks; see for example, U.S. Patent Nos.
  • Preferred MEKK protein for use with the method includes recombinant MEKK protein. More preferred MEKK protein includes at least a portion of a MEKK protein having a kinase domain or apoptotic domain of MEKK.
  • kits to identify compounds capable of regulating signals initiated from a receptor on the surface of a cell, such signals involving in some respect, MEKK protein.
  • kits include: (a) at least one cell containing MEKK protein; (b) a ligand capable of binding to a receptor on the surface of the cell; and (c) a means for assessing the ability of a putative regulatory compound to - 42 -
  • Such a means for detecting phosphorylation include methods and reagents known to those of skill in the art, for example, phosphorylation can be detected using antibodies specific for phosphorylated amino acid residues, such as tyrosine, serine and threonine.
  • phosphorylation can be detected using antibodies specific for phosphorylated amino acid residues, such as tyrosine, serine and threonine.
  • kit one is capable of determining, with a fair degree of specificity, the location along a signal transduction pathway of particular pathway constituents, as well as the identity of the constituents involved in such pathway, at or near the site of regulation.
  • a kit of the present invention can include: (a) MEKK protein; (b) MEKK substrate, such as MEK; and (c) a means for assessing the ability of a putative inhibitory compound to inhibit phosphorylation of the MEKK substrate by the MEKK protein.
  • a mammalian MEKK gene can be used to rescue a yeast cell having a defective stel l (or byr2) gene, such as a temperature sensitive mutant stel l mutant (cfi, Francois et al. (1991) J Biol Chem 266:6174-80; and Jenness et al (1983) Cell 35:521-9).
  • a humanized yeast can be generated by amplifying the coding sequence of the human MEKK clone, and subcloning this sequence into a vector which contains a yeast promoter and termination sequences flanking the MEKK coding sequences. This plasmid can then be used to transform an stel l ⁇ s mutant.
  • cultures of the transformed cells can be grown at an permissive temperature for the TS mutant. Turbidity measurements, for example, can be used to easily determine the growth rate. At the non-permissive temperature, pheromone responsiveness of the yeast cells becomes dependent upon expression of the human MEKK protein. Accordingly, the humanized yeast cells can be utilized to identify compounds which inhibit the action of the human MEKK protein. It is also deemed to be within the scope of this invention that the humanized yeast cells of the present assay can be generated so as to comprise other human cell-cycle proteins. For example, human MEK and human MAPK can also be expressed in the yeast cell in place of ste7 and Fus3/Kssl . In this manner, the reagent cells of the present assay can be generated to more closely approximate the natural interactions which the mammalian MEKK protein might experience.
  • certain formats of the subject assays can be used to identify drugs which inhibit proliferation of yeast cells or other lower eukaryotes, but which have a substantially reduced effect on mammalian cells, thereby improving therapeutic index of the drug as an anti-mycotic agent.
  • the identification of such compounds is made possible by the use of differential screening assays which detect and compare drug-mediated disruption of binding between two or more different types of MEKK/MEKK-bp complexes, or which differentially inhibit the kinase activity of, for example, stel 1 relative to a mammalian MEKK.
  • Differential screening assays can be used to exploit the difference in drug-mediated disruption of human MEKK complexes and yeast stel l/byr2 complexes in order to identify agents which display a statistically significant increase in specificity for disrupting the yeast complexes (or kinase activity) relative to the human complexes.
  • lead compounds which act specifically to inhibit proliferation of pathogens such as fungus involved in mycotic infections, can be developed.
  • the present assays can be used to screen for agents which may ultimately be useful for inhibiting at least one fungus implicated in such mycosis as candidiasis, aspergillosis, mucomycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para- actinomycosis, penicilliosis, monoliasis, or sporotrichosis.
  • mycosis as candidiasis, aspergillosis, mucomycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis,
  • the present assay can comprise comparing the relative effectiveness of a test compound on mediating disruption of a human MEKK with its effectiveness towards disrupting the equivalent stel l/byr2 kinase from genes cloned from yeast selected from the group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida par apsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, or Candida rugosa.
  • the present assay can be used to identify anti-fungal agents which may have therapeutic value in the treatment of aspergillosis by making use of genes cloned from yeast such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus.
  • yeast such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus.
  • the complexes can be derived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, or Mucor pusillus.
  • Sources of other stel l/byr2 homologs for comparison with a human MEKK includes the pathogen Pneumocystis carinii.
  • Another aspect of the present invention relates to the treatment of an animal having a medical disorder that is subject to regulation or cure by manipulating a signal transduction pathway in a cell involved in the disorder.
  • medical disorders include disorders which result from abnormal cellular growth or abnormal production of secreted cellular products.
  • medical disorders include, but are not limited to, cancer, autoimmune disease, inflammatory responses, allergic responses and neuronal disorders, such as Parkinson's disease and Alzheimer's disease.
  • Preferred cancers subject to treatment using a method of the present invention include, but are not limited to, small cell carcinomas, non-small cell lung carcinomas with overexpressed EGF receptors, breast cancers with overexpressed EGF or Neu receptors, tumors having overexpressed growth factor receptors of established autocrine loops and tumors having overexpressed growth factor receptors of established paracrine loops.
  • small cell carcinomas non-small cell lung carcinomas with overexpressed EGF receptors
  • breast cancers with overexpressed EGF or Neu receptors tumors having overexpressed growth factor receptors of established autocrine loops and tumors having overexpressed growth factor receptors of established paracrine loops.
  • the term treatment can refer to the regulation of the progression of a medical disorder or the complete removal of a medical disorder (e.g., cure).
  • Treatment of a medical disorder can comprise regulating the signal transduction activity of a cell in such a manner that a cell involved in the medical disorder no longer responds to extracellular stimuli (e.g., growth factors or cytokines), or the killing of a cell involved in the medical disorder through cellular apoptosis.
  • extracellular stimuli e.g., growth factors or cytokines
  • the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival, and/or promoting (or alternatively inhibiting) proliferation of a cell responsive to a growth factor, morphogen or other environmental cue which effects the cell through at least one signal transduction pathway which includes a MEKK protein.
  • the method comprises contacting the cells with an amount of an agent which significantly (statistical) modulates MEKK-dependent signaling by the factor.
  • a "MEKK therapeutic,” whether inductive or anti-inductive with respect to signaling by a MEKK-dependent pathway, can be, as appropriate, any of the preparations described above, including isolated polypeptides, gene therapy constructs, antisense molecules, peptidomimetics or agents identified in the drug assays provided herein.
  • MEKK therapeutics of the present invention can provide therapeutic benefits where the general strategy being the inhibition of an anomalous cell proliferation.
  • Diseases that might benefit from this methodology include, but are not limited to various cancers and leukemias, psoriasis, bone diseases, fibroproliferative disorders such as involving connective tissues, atherosclerosis and other smooth muscle proliferative disorders, as well as chronic inflammation.
  • the present invention contemplates the use of MEKK therapeutics for the treatment of differentiative disorders which result from, for example, de-differentiation of tissue which may (optionally) be accompanied by abortive reentry into mitosis, e.g., apoptosis.
  • degenerative disorders include chronic neurodegenerative diseases of the nervous system, including Alzheimer's disease, Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations.
  • Other differentiative disorders include, for example, disorders associated with connective tissue, such as may occur due to de- - 45 -
  • MEKK agonists and antagonists can be employed in a differential manner to regulate different stages of organ repair after physical, chemical or pathological insult.
  • such regimens can be utilized in repair of cartilage, increasing bone density, liver repair subsequent to a partial hepatectomy, or to promote regeneration of lung tissue in the treatment of emphysema.
  • Another aspect of the present invention concerns transgenic animals which are comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express an exogenous MEKK protein in one or more cells in the animal.
  • a MEKK transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.
  • the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern.
  • mosaic expression of a MEKK protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, lack of MEKK expression which might grossly alter development in small patches of tissue within an otherwise normal embryo.
  • tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.
  • temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
  • target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
  • the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing - 46 -
  • Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject MEKK proteins.
  • excision of a target sequence which interferes with the expression of a recombinant MEKK gene such as one which encodes an antagonistic homolog or an antisense transcript, can be designed to activate expression of that gene.
  • This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the MEKK gene from the promoter element or an internal stop codon.
  • the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element.
  • inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.
  • either the crelloxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.
  • Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences.
  • loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
  • the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
  • genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
  • Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
  • the activation expression of a recombinant MEKK protein can be regulated via control of recombinase expression.
  • MEKK protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both - 47 -
  • the Cre recombinase and a recombinant MEKK gene can be provided through the construction of "double" transgenic animals.
  • a convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., a MEKK gene and recombinase gene.
  • One advantage derived from initially constructing transgenic animals containing a MEKK transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein, whether agonistic or antagonistic, can be deleterious upon expression in the transgenic animal. In such an instance, a founder population, in which the subject transgene is silent in all tissues, can be propagated and maintained.
  • founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues and/or a desired temporal pattern.
  • an antagonistic MEKK transgene is silent will allow the study of progeny from that founder in which disruption of MEKK mediated induction in a particular tissue or at certain developmental stages would result in, for example, a lethal phenotype.
  • prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the MEKK transgene.
  • Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
  • conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g., a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner.
  • a MEKK transgene could remain silent into adulthood until "turned on” by the introduction of the trans- activator.
  • the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
  • Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell.
  • the zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution.
  • zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient - 48 -
  • Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
  • Retroviral infection can also be used to introduce MEKK transgenes into a non- human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
  • the viral vector system used to introduce the transgene is typically a replication- defective retrovirus carrying the transgene (Jahner et al.
  • the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
  • transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.
  • Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • Jaenisch, R. (1988) Science 240:1468-1474 For review see Jaenisch, R. (1988) Science 240:1468-1474.
  • MEKK knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g., by homologous recombination to insert recombinase target sequences flanking portions of an endogenous MEKK gene, such - 49 -
  • tissue specific and/or temporal control of inactivation of a MEKK allele can be controlled as above.
  • an isolated, or biologically pure, peptide is a peptide that has been removed from its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the protein has been purified.
  • An isolated compound of the present invention can be obtained from a natural source or produced using recombinant DNA technology or chemical synthesis.
  • an isolated peptide can be a full-length protein or any homolog of such a protein in which amino acids have been deleted (e.g., a truncated version of the protein), inserted, inverted, substituted and/or derivatized (e.g.
  • a “mimetope” refers to any compound that is able to mimic the ability of an isolated compound of the present invention.
  • a mimetope can be a peptide that has been modified to decrease its susceptibility to degradation but that still retain regulatory activity.
  • Other examples of mimetopes include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof.
  • a mimetope can be obtained by, for example, screening libraries of natural and synthetic compounds as disclosed herein that are capable of inhibiting the binding of Ras superfamily protein to MEKK.
  • a mimetope can also be obtained by, for example, rational drug design.
  • the three-dimensional structure of a compound of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography.
  • NMR nuclear magnetic resonance
  • the three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modeling.
  • the predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi).
  • the therapeutic methods of the present invention may also comprise injecting an area of a subject's body with an effective amount of a naked plasmid DNA compound (such as is taught, for example in Wolff et ⁇ l, 1990, Science 247, 1465-1468).
  • a naked plasmid DNA compound comprises a nucleic acid molecule encoding a MEKK protein of the present invention, operatively linked to a naked plasmid DNA vector capable of being taken up by and expressed in a recipient cell located in the body area.
  • naked plasmid DNA compound of the present invention comprises a nucleic acid molecule encoding a truncated MEKK protein having deregulated kinase activity.
  • Preferred naked plasmid DNA vectors of the present invention include those known in the art.
  • a naked plasmid DNA compound of the present invention transforms cells within the subject and directs the production of at least a portion of a MEKK protein or RNA nucleic acid molecule that is capable of regulating the apoptosis of the cell.
  • a naked plasmid DNA compound of the present invention is capable of treating a subject suffering from a medical disorder including cancer, autoimmune disease, inflammatory responses, allergic responses and neuronal disorders, such as Parkinson's disease and Alzheimer's disease.
  • a naked plasmid DNA compound can be administered as an anti -tumor therapy by injecting an effective amount of the plasmid directly into a tumor so that the plasmid is taken up and expressed by a tumor cell, thereby killing the tumor cell.
  • an effective amount of a naked plasmid DNA to administer to a subject comprises an amount needed to regulate or cure a medical disorder the naked plasmid DNA is intended to treat, such mode of administration, number of doses and frequency of dose capable of being decided upon, in any given situation, by one of skill in the art without resorting to undue experimentation.
  • An isolated compound of the present invention can be used to formulate a therapeutic composition.
  • a therapeutic composition of the present invention includes at least one isolated peptide of the present invention.
  • a therapeutic composition for use with a treatment method of the present invention can further comprise suitable excipients.
  • a therapeutic compound for use with a treatment method of the present invention can be formulated in an excipient that the subject to be treated can tolerate.
  • excipients examples include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful excipients include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers examples include phosphate buffer, bicarbonate buffer and Tris buffer
  • preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection.
  • the excipient in a non-liquid formulation, can comprise - 51 -
  • dextrose human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • a therapeutic compound for use with a treatment method of the present invention can also comprise a carrier.
  • Carriers are typically compounds that increase the half-life of a therapeutic compound in the treated animal. Suitable carriers include, but are not limited to, liposomes, micelles, cells, polymeric controlled release formulations, biodegradable implants, bacteria, viruses, oils, esters, and glycols. Preferred carriers include liposomes and micelles.
  • a therapeutic compound for use with a treatment method of the present invention can be administered to any subject having a medical disorder as herein described.
  • Acceptable protocols by which to administer therapeutic compounds of the present invention in an effective manner can vary according to individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art without resorting to undue experimentation.
  • An effective dose refers to a dose capable of treating a subject for a medical disorder as described herein. Effective doses can vary depending upon, for example, the therapeutic compound used, the medical disorder being treated, and the size and type of the recipient animal.
  • Effective doses to treat a subject include doses administered over time that are capable of regulating the activity, including growth, of cells involved in a medical disorder.
  • a first dose of a naked plasmid DNA compound of the present invention can comprise an amount that causes a tumor to decrease in size by about 10% over 7 days when administered to a subject having a tumor.
  • a second dose can comprise at least the same the same therapeutic compound than the first dose.
  • Another aspect of the present invention includes a method for prescribing treatment for subjects having a medical disorder as described herein.
  • a preferred method for prescribing treatment comprises: (a) measuring the MEKK protein activity in a cell involved in the medical disorder to determine if the cell is susceptible to treatment using a method of the present invention; and (b) prescribing treatment comprising regulating the activity of a MEKK-dependent pathway relative to the activity of a Raf- dependent pathway in the cell to induce the apoptosis of the cell.
  • the step of measuring MEKK protein activity can comprise: (1) removing a sample of cells from a subject; (2) stimulating the cells with a TNF ⁇ ; and (3) detecting the state of phosphorylation of MKK3, MKK4 or JNKK protein using an immunoassay using antibodies specific for phosphothreonine -and/or phosphoserine.
  • the present invention also includes antibodies capable of selectively binding to a MEKK protein of the present invention.
  • Such an antibody is herein referred to as an anti-MEKK antibody.
  • Polyclonal populations of anti-MEKK antibodies can be contained in a MEKK antiserum.
  • MEKK antiserum can refer to affinity purified polyclonal antibodies, ammonium sulfate cut antiserum or whole antiserum.
  • selective binds to refers to the ability of such an antibody to preferentially bind to MEKK proteins.
  • Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, enzyme immunoassays (e.g., ELISA), radioimmunoassays, immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.
  • Antibodies of the present invention can be either polyclonal or monoclonal antibodies and can be prepared using techniques standard in the art.
  • Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein used to obtain the antibodies.
  • antibodies are raised in response to proteins that are encoded, at least in part, by a MEKK nucleic acid molecule. More preferably antibodies are raised in response to at least a portion of a MEKK protein, and even more preferably antibodies are raised in response to either the amino terminus or the carboxyl terminus of a MEKK protein.
  • an antibody of the present invention has a single site binding affinity of from about 10 3 M _1 to about 10 12 M _1 for a MEKK protein of the present invention.
  • a preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of a MEKK protein to produce the antibody and recovering the antibodies.
  • Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used to identify unique MEKK proteins and recover MEKK proteins.
  • Another aspect of the present invention comprises a therapeutic compound capable of regulating the activity of a MEKK-dependent pathway in a cell identified by a process, comprising: (a) contacting a cell with a putative regulatory molecule; and (b) determining the ability of the putative regulatory compound to regulate the activity of a MEKK-dependent pathway in the cell by measuring the activation of at least one member of said MEKK-dependent pathway.
  • Preferred methods to measure the activation of a member of a MEKK-dependent pathway include measuring the transcription regulation activity of c-Myc protein, measuring the phosphorylation of a protein selected from the group consisting of MEKK, JNKK, JNK, Jun, ATF-2, Myc, and combinations thereof.
  • the partial murine MEKKl nucleotide sequence is shown in SEQ ID NO: 1.
  • the predicted amino acid sequence is shown in SEQ ID NO2. Additional cloning based on the sequence of the partial murine MEKKl shown in SEQ ID NO:l resulted in the nucleotide sequence of a full-length murine MEKKl DNA which is set forth in Figure 2 and as SEQ ID NO:3.
  • the predicted amino acid sequence of full-length murine MEKKl is set forth as SEQ ID NO:4.
  • PCR Amplification The sense strand primer 5'-GAACACCATCCAGAAGTTTG-3' (SEQ ID NO: 13), which was designed from the mouse MEKKl (mMEKKl) cDNA sequence, was used in conjunction with the antisense primer 5'-CACTTTGTAGACAGGGTCAGC-3' (SEQ ID NO: 14) in a polymerase chain reaction (PCR) using the first strand cDNA described above as a template (RT-PCR) to amplify the region from bases 1211-1950.
  • PCR polymerase chain reaction
  • RT-PCR polymerase chain reaction
  • Taq DNA Polymerase Boehringer Mannheim
  • the resulting sequence was aligned to the known mMEKKl sequence, and determined to be hMEKKl by homology.
  • the sense primer 5'-TGGGTCGCCTCTGTCTTATAGACAG-3' (SEQ ID NO: 15) was used in conjunction with the antisense primer 5'- CACATCCTGTGCTTGGTAAC-3' (SEQ ID NO:16) in a RT-PCR of 30 cycles (1 min. 94°C; 1 min., 50°C; 2 min., 72°C), followed by a 10 min. incubation at 72°C.
  • a band of approximately 1.5 kb was isolated by purification from a 1% agarose gel, ligated, cloned, and sequenced as stated above.
  • the sense primer 5'-AGGACAAGTGCAGGTTAGATG-3' was used in a RT-PCR of 30 cycles (1 min., 94°C; 1 min., 50°C; 2 min., 72°C), followed by a 10 min. incubation at 72°C.
  • a band of approximately 1.3 kb was isolated by purification from a 1% agarose gel, ligated, cloned, and sequenced as stated above. Sequence was also confirmed for - 55 -
  • the sense primer 5'- CGGCCTGGAAGCACGAGTGGT-3' (SEQ ID NO: 19) was used in conjunction with the antisense primer 5'-TTCATCCTTGATGCTGTTTTC-3' (SEQ ID NO:20) in a RT-PCR of 30 cycles (1 min., 94°C; 1 min., 50°C; 2 min., 72°C), followed by a 10 min. incubation at 72°C.
  • a band of approximately 700 bp was isolated by purification from a 1 % agarose gel, ligated, cloned, and sequenced as stated above. The overlapping sequence data was compiled into a single contig using Sequencer 2.0 (Gene Codes), and aligned to the mMEKKl sequence.
  • Sequencer 2.0 Gene Codes
  • a BLAST search using the amino acid sequences of murine MEKKl and human MEKKl as described in this example reveals nucleotide and amino acid sequences having substantial homology to those set forth in SEQ ID NOs:3-6 (e.g., sequences having Accession No. 423499, Accession No. 2507203 and Accession No. U23470).
  • Example 2 Apoptotic Fragments of MEKKl
  • MEK kinase 1 MEKKl
  • Asp 8 ' 4 Cleavage of mouse MEKKl at Asp 8 ' 4 generates a 91 kDa kinase fragment and a 113 kDa NH2-terminal fragment.
  • the kinase fragment of ⁇ MEKKl induces apoptosis.
  • Cleavage of MEKKl and apoptosis are inhibited by p35 and CrmA, viral inhibitors of the ICE/FLICE proteases that commit cells to apoptosis.
  • MEKKl sequence 871 DTVD 874 (SEQ ID NO: 7), a cleavage site for CCP32-like proteases, to alanines inhibited proteolysis of MEKKl and apoptosis induced by overexpression of MEKKl .
  • Inhibition of MEKKl proteolysis inhibited apoptosis but did not block MEKKl stimulation of c-Jun kinase activity, indicating that c-Jun kinase activation was not sufficient for apoptosis.
  • Apoptosis or programmed cell death is a physiological process important in differentiation and tissue modeling (Williams and Smith, 1993; adherer, 1995). Apoptosis can be triggered by many different stimuli including growth factor deprivation (Xia et al, 1995; Park et al, 1996), exposure of specific cell types to cytokines such as TNF ⁇ and Fas ligand (Vandenabeele et al, 1995; Kagi et al, 1994; Lowin et al, 1994), virus - 56 -
  • ICE/FLICE substrates have been identified including poly (ADP-ribose) polymerase (Lazebnik et al, 1994), UI small nuclear ribonucleoprotein (Casciola-Rosen et al, 1994), lamin (Lazebnik et al, 1995), D4-GDI (Na et al, 1996), fodrin (Cryns et al, 1996), protein kinase C ⁇ (Emoto et al, 1995), sterol regulatory element binding protein (Wang et al, 1996), retinoblastoma protein (An and Dou, 1996), DNA-dependent protein kinase (Casciola-Rosen et al , 1995), and the proteases themselves (Orth et al, 1996).
  • poly (ADP-ribose) polymerase Lazebnik et al, 1994
  • UI small nuclear ribonucleoprotein Casciola-Ros
  • ICE-like proteases such as Ced-3 have a specificity for proteins encoding the four amino acid sequence YVAD (SEQ ID NO: 10) (Howard et al, 1991) while CPP32-like proteases have a preference for the sequence DEVD (SEQ ID NO: 11) (Nicholson et al, 1995). Both groups of proteases cleave at the terminal aspartic acid residue of the recognition sequence.
  • Several viruses encode proteins that are specific inhibitors of the ICE/FLICE proteases.
  • CrmA is a poxvirus protein that inhibits ICE-like proteases
  • p35 is a baculovirus protein that has broad inhibitory activity to ICE/FLICE-like proteases (Fraser and Evan, 1996; Clem et al, 1996).
  • Expression of CrmA and p35 inhibit the apoptotic response to many different stimuli demonstrating the requirement of ICE/FLICE proteases during programmed cell death (Beidler et al, 1996; Los et al, 1995).
  • JNK c-Jun kinases
  • p38 p38 kinases
  • MEKKs protein serine-threonine kinases
  • MEKKl has been found to have the unique property of being a strong stimulator of apoptosis (Lassignal Johnson et al, 1996; Xia et al, 1995). The other MEKKs, even though they all activate c-Jun kinases and ERKs to different levels, do not induce apoptosis, suggesting MEKKl has unique substrates that mediate the death response.
  • the kinase domain of MEKKl is only 50%> conserved relative to the kinase domains of MEKK 2, 3 and 4, consistent with MEKKl having unique substrate recognition properties and catalytic activity involved in mediating the apoptotic response.
  • MEKKl is a 196 kDa protein that encodes a protease cleavage sequence for CPP32-like proteases. None of the other MEKKs or known kinases that regulate MAPK pathways have a consensus ICE/FLICE cleavage site.
  • MEKKl is a substrate for proteases inhibited by the p35 baculovirus protein. When the kinase domain is released from the holo-MEKKl protein it functions as a physiological activator of apoptosis. UV irradiation and DNA damaging chemicals activate MEKKl kinase activity and induce its proteolytic cleavage indicating that MEKKl contributes to apoptosis in response to environmental stresses.
  • HEK293 Human embryonal kidney 293 cells (HEK293) stably expressing the EBNA-1 protein from Epstein-Barr virus (Invitrogen) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin/streptomycin and containing 10% bovine calf serum (BCS). The cells were transfected using lipofectamine (Gibco).
  • DMEM Dulbecco's modified Eagle's medium
  • BCS bovine calf serum
  • the full length cD A encoding mouse MEKKl was modified by addition of the HA-tag sequence (MGYPYDVDYAS) (SEQ ID NO: 12) at its NH 2 -terminus and inserted into the expression plasmid pCEP4 (Invitrogen), resulting in plasmid MEKKl .cp4.
  • the MEKKl sequences DTVD (amino acids 871-874) and DEVE (amino acids 857-860) in MEKKl.cp4 were substituted with alanines using a PCR strategy.
  • the resulting plasmids were named DTVD_A.cp4 and DEVE_A.cp4.
  • Plasmid pCDNA_3.cp4 is the result of the ligation of pCEP4 and pCDNA-3. - 58 -
  • Lysis buffer 70 mM ⁇ -glycerophosphate, 1 mM EGTA, 100 ⁇ M Na 3 VO 4 , 1 mM DTT, 2 mM MgCl2, 0.5% Triton-XlOO, 20 ⁇ g/ml aprotinin
  • aprotinin 0.5% Triton-XlOO, 20 ⁇ g/ml aprotinin
  • JNK activity was measured using a solid phase kinase assay in which glutathione S-transferase-c-Junn _ ⁇ g (GST- Jun) bound to glutathione-Sepharose 4B beads was used to affinity-purify JNK from cell lysates as described (Gardner and Johnson, 1996; Hibi et al, 1993).
  • JNK1 or JNK2 were immunoprecipitated with isoform specific antibodies (Santa Cruz Biotechnology) and GST- Jun used as substrate in an in vitro kinase assay (Hibi et al, 1993). Quantitation of the phosphorylation of GST- Jun was performed with a Phosphorlmager.
  • ERK2 was immunoprecipitated as described above for the JNK isoforms using the
  • ERK2 (C-14) antibody (Santa Cruz Biotechnology).
  • the beads were washed twice with 1 ml lysis buffer and twice with 1 ml lysis buffer without Triton-XlOO. Thirty-five ⁇ l of the last wash was left in the tube and mixed with 20 ⁇ l of kinase 2X mix (50 mM ⁇ - glycerophosphate, 100 ⁇ M Na 3 VO 4 , 20 mM MgCl 2 , 200 ⁇ M ATP, 1 ⁇ Ci/ ⁇ l ⁇ 32 P- ATP, 400 ⁇ M EGF receptor peptide 662-681, 100 ⁇ g/ ⁇ l IP-20, 2 mM EGTA), incubated 20 min at 20°C and spotted on P81 Whatman paper. The samples were washed thrice for 5 min each in 75 mM phosphoric acid and once for 2 min in acetone, air-dried, and their radioactivity determined in a ⁇ counter.
  • MEKKl was immunoprecipitated from cell lysates (200-500 ⁇ g) with the antibodies raised against specific sequences of MEKKl or the 12CA5 antibody that recognizes the HA-tag sequence.
  • the immunoprecipitates were used in an in vitro kinase assay with recombinant kinase inactive SEK1 (SEK1 K-»M) as previously described (Blank et al, 1996). - 59 -
  • Cells were grown on glass coverslips and transfected using lipofectamine. Two days after transfection, the medium was removed and the cells were fixed in 2% paraformaldehyde, 3% sucrose in phosphate buffered saline (PBS) for 10 min at room temperature. Following three washes with PBS, the cells were permeabilized for 10 min with 2% Triton-XlOO in PBS. After three PBS washes, the cells were blocked with filtered cultured medium for 15 min.
  • PBS phosphate buffered saline
  • the coverslips were then incubated 1 hour in TdT reaction mix (200 mM potassium cacodylate, 25 mM Tris ⁇ Cl, pH 6.6, 250 ⁇ g/ml BSA, 5 mM CoCl 2 , 0.25 U/ ⁇ l TdT [Boehringer], 10 ⁇ M biotin-dUTP [Boehringer]) at 37 °C in a humidified atmosphere.
  • TdT reaction mix 200 mM potassium cacodylate, 25 mM Tris ⁇ Cl, pH 6.6, 250 ⁇ g/ml BSA, 5 mM CoCl 2 , 0.25 U/ ⁇ l TdT [Boehringer], 10 ⁇ M biotin-dUTP [Boehringer]
  • the coverslips were incubated for 1 hour at room temperature with a 1/500 dilution in filtered culture medium of an affinity purified rabbit antisera directed at the peptide DRPPSRELLKHPVFR of mouse MEKKl (amino acids 1476-1490) (Lange-Carter et al, 1993).
  • the coverslips were then washed 6x over a 30 min period with PBS and incubated 1 hour at room temperature with a 1/1000 dilution in filtered culture medium of a donkey anti-rabbit, Cy->- conjugated, antibody (Jackson Immunological) mixed with 5 ⁇ g/ml streptavidin conjugated with FITC (Jackson Immunological).
  • the coverslips were washed 6x with PBS and incubated overnight in PBS before being mounted in 20 mg/ml o-phenyldiamine-diHCl (Sigma) in 0.1 M Tris pH 8.5, 90% glycerol. Images were taken using a Leica DMRXA microscope and analyzed with the SlideBook v2.0 software (Intelligent Imaging Innovations, Denver). The subcellular localization of endogenous MEKKl observed with the anti-COOH-terminal MEKKl antibody was identical to that observed with a second antibody recognizing the NH2-terminal portion of the MEKKl protein.
  • MEKKl was immunoprecipitated from cell lysates (200-500 ⁇ g) using the 96-001 (NH 2 ) antisera, washed twice with 1 ml extraction buffer (BE) [1% Triton-XlOO; 10 mM Tris pH 7.4; 50 mM Nalco; 50 mM AF; 5 mM EDTA], twice with 1 ml CT (50 mM Tris pH 7.0; 0.1 mM call 2 ) and once with 1 ml CT containing 60 mM ⁇ - mercaptoethanol, 1 mM MgCl 2 . 35 ⁇ l of the last wash was left in the tube and 0.5 U of PP-2A (Upstate Biotechnology) was added for 30-45 min.
  • PP-2A Upstate Biotechnology
  • the phosphatase reaction was terminated by adding 1 ⁇ l of 200 mM Na3VU4.
  • the immunoprecipitates were washed three more times with 1 ml PAN (10 mM PIPES; 100 mM NaCl; 20 ⁇ g/ml aprotinin) before being mixed with the SEK1 K(M substrate and ⁇ 32 P-ATP.
  • HEK293 cells were transfected with a plasmid encoding the mouse MEKKl and stained 2 days later for MEKKl expression using an antibody directed at the COOH-terminus of the protein.
  • DNA fragmentation a feature often associated with apoptosis, was measured by terminal-deoxy-transferase-mediated incorporation of fluorescent dUTP. A large proportion of HEK293 cells expressing MEKKl had fragmented DNA.
  • the MEKKl expressing cells characteristically rounded up and began to lift off the coverslips.
  • MEKKl also induced chromatin condensation and the nuclei in these cells often dissociated from the surrounding cytoplasm.
  • Quantitation of cells exhibiting DNA fragmentation and cells expressing MEKKl revealed that about 30%> of MEKKl - expressing cells were apoptotic after 48 hr. This is an underestimate because the apoptotic cells eventually detach from the coverslips and often loose their nucleus.
  • the kinase activity of MEKKl is required for the induction of cell death (Lassignal Johnson et al, 1996). - 61 -
  • MEKK1 -induced DNA fragmentation is inhibited by p35 and CrmA.
  • CrmA and p35 inhibit cleavage of the 196 kDa MEKKl protein and generation of an activated kinase fragment.
  • lysates from cells transfected with HA- tagged MEKKl alone or in combination with CrmA or p35 were used for immunoprecipitation with the 12CA5 HA antibody or with an antibody specific for the COOH-terminal moiety of MEKKl (antibody 95-012).
  • the immunoprecipitates were then incubated with a MEKKl substrate (SEKl K(M) and ⁇ 32 P-ATP.
  • SEKl K(M) ⁇ 32 P-ATP
  • p35 inhibited cleavage occurs at position Asp- ⁇ in the mouse MEKKl protein.
  • the p35-inhibited cleavage of MEKKl generates a COOH-terminal fragment of about 90 kDa and a NH 2 -terminal fragment of about 110 kDa, indicating that the - 63 -
  • cleavage occurs between residues 820-900.
  • Two tetrapeptide sequences that are found in this region of MEKKl closely resemble the CPP32 cleavage site, DEVD (SEQ ID NO: 12) (Nicholson et al, 1995). These sequences are 857 DEVE 860 (SEQ ID NO: 7) and 8 lDTVD 874 (SEQ ID NO: 8) (see Fig. 4).
  • proteases inhibited by p35 have been shown to be cysteine proteases cleaving after the aspartic acid residue in the fourth position of the consensus cleavage sequence (Nicholson et al, 1995; Howard et al, 1991) and, therefore only the DTVD (SEQ ID NO: 8) sequence should be a cleavage site for the CPP32-like protease.
  • Two mutants were generated that have either the DEVE (SEQ ID NO: 7) or the DTVD (SEQ ID NO: 8) sequence replaced with alanine residues (see Fig. 4).
  • the kinase activity of the mutants expressed in HEK293 cells was determined. Immunoprecipitating full length 196 kDa MEKKl or mutant MEKKl proteins with the 12CA5 antibody resulted in similar SEKl phosphorylating activities. However, when the antibodies directed towards the COOH-terminus of the protein were used, SEKl phosphorylating activity was reduced in DTVD— >A expressing cells as compared to the activity found in wild-type or DEVE- A expressing cells. The reduced kinase activity was comparable to the basal SEKl phosphorylating activity observed when the full length proteins were immunoprecipitated.
  • the mutant DTVD-»A MEKKl protein has a low but measurable kinase activity towards SEKl because fragment C is not generated. The same result was observed when the cleavage of MEKKl into fragments B and C was inhibited by p35 expression.
  • Fig. 5 describes a model of the MEKKl cleavage events occurring in transfected cells.
  • overexpression of MEKKl induces deregulated cleavage events generating two sets of fragments (A and D; B and C).
  • Fragment C encoding the catalytic domain of MEKKl has a stronger kinase activity than the full length protein.
  • Proteases of the ICE/FLICE family are responsible for the cleavage of MEKKl into fragments B and C because this cleavage can be inhibited by p35 and CrmA.
  • Mutagenesis experiments revealed that the cleavage site generating fragments B and C is DTVD 874 (SEQ ID NO: 8). Fragment C can be further processed into a smaller polypeptide (fragment D) which may be rapidly degraded. It is possible - 64 -
  • proteolytic activity which generates fragment D is part of a regulatory mechanism involved in the termination of the response induced by cleavage of MEKKl into the active fragment C.
  • the DTVD— >A mutant has a reduced ability to promote DNA fragmentation in HEK293 cells.
  • DTVD— » A mutant induces DNA fragmentation when expressed in HEK293 cells.
  • Expression of the DEVE ⁇ A mutant or the wild-type MEKKl protein induced DNA fragmentation.
  • cells expressing the DTVD ⁇ A mutant MEKKl protein showed little DNA fragmentation. Quantitation of the response revealed that the number of DTVD ⁇ A expressing cells that showed some DNA fragmentation was reduced by 65% compared to the cells transfected with wild-type MEKKl or the DEVE ⁇ A mutant. This indicates that cleavage of MEKKl into fragments B and C is required to induce cell death.
  • p35 inhibits ⁇ MEKK1 -induced apoptosis.
  • the 37 kDa kinase domain of MEKKl ( ⁇ MEKK1) is a strong inducer of apoptosis (Lassignal Johnson et al, 1996; Xia et al, 1995). Since p35 inhibits programmed cell death induced by most, if not all, apoptotic stimuli (Clem et al, 1996), it was also determined whether this inhibitor could also block ⁇ MEKK1 -induced apoptosis. ⁇ MEKK1 induced DNA fragmentation when expressed in HEK293 cells. This effect was inhibited by co-expression of p35.
  • ⁇ MEKK1 and p35 were co-expressed. Even if the co-transfected cells showed less DNA fragmentation compared to the cells transfected with ⁇ MEKK1 alone, they were clearly affected by the expression of ⁇ MEKK1 and were rounded and most showed some membrane blebbing. This differed from the effect of p35 in full length MEKKl - transfected cells, where the inhibitor appeared to better protect the cells from DNA fragmentation and obvious morphological changes, the predicted result if cleavage of MEKKl results in the release of an activated kinase domain.
  • MEKKl activates the ERK and JNK pathways (Xu et al, 1996). Since activation of the JNK pathway has been proposed to induce apoptosis (Verheij et al, 1996), it was next determined whether inhibitory mutants of JNKl or JNK2 (JNKl -APF and JNK2-APF, respectively) could prevent MEKKl -induced DNA fragmentation. While JNKl -APF had no protective effect, JNK2-APF slightly (by about 30%) reduced the number of MEKKl -expressing apoptotic cells.
  • the competitive inhibitory JNK mutants had no effect on the generation of any cleavage products, indicating that the JNK2-APF-mediated inhibition of MEKKl -induced DNA fragmentation is not related to the cleavage of MEKKl.
  • Activation of ERK2 or the JNKs by MEKKl was unaffected by the co-expression of JNK1-APF, JNK2-APF, p35 or CrmA.
  • specific JNK isoforms were immunoprecipitated, only JNKl -APF and JNK2-APF partially inhibited JNKl and JNK2 activity, respectively. The partial inhibition may be due to cross-reactivity of the antibodies used (Gupta et al, 1996).
  • the DEVE ⁇ A and DTVD ⁇ A mutants activated JNK to the same level as wild type MEKKl .
  • Transfection of MEKKl in HEK293 cells did not activate the p38 kinase. Cumulatively, these results show that in conditions where MEKKl -induced DNA fragmentation is inhibited (i.e. when p35 is cotransfected with MEKKl or when the DTVD ⁇ A mutant is expressed), the ERK and the JNK pathways are still activated to an extent similar to that found in MEKKl -transfected cells. This indicates that neither the ERK nor the JNK pathways are sufficient to promote or inhibit the cell death pathway induced by cleavage of MEKKl.
  • UV irradiation of HEK293 cells induces a rapid phosphorylation and subsequent cleavage of the endogenous MEKKl protein.
  • a time course was performed to determine the effects of UV irradiation on the endogenous MEKKl protein, activation of the JNK pathway and the extent of apoptosis resulting from the exposure of the cells to a stress stimulus.
  • 15 min after UV irradiation an MEKKl species is generated that was upward gel-shifted compared to the MEKKl species detected before exposure to UV irradiation.
  • One hour after irradiation most of the full length MEKKl protein was upward gel-shifted.
  • Eight hours after irradiation the amount of the gel-shifted MEKKl started to decrease and 20 hours after UV treatment only a trace amount of full length MEKKl was detected.
  • the MEKKl fragment detected by the 96-001 (NH2) antibody was barely seen in the control condition.
  • Cleavage of MEKKl can be mediated by different stress stimuli.
  • the ERK pathway has been shown to have a protective response against an apoptotic stimulus in a few cell types (Xia et al, 1995; Gardner and Johnson, 1996).
  • discordance for a role of c-Jun kinases and p38 kinases in mediating apoptosis also exists.
  • MEKKl mediated apoptosis was shown to be independent of c-Jun kinase activation (Lassignal Johnson et al, 1996).
  • a similar separation of c-Jun kinase activation and apoptosis was observed with the TNF receptor (Liu et al, 1996b).
  • JNK pathway is clearly not sufficient to induce the apoptosis mediated by MEKKl .
  • MEKK1 -mediated apoptosis requires both kinase activity and proteolytic cleavage.
  • the sequence in the rat MEKKl sequence that corresponds to the murine DTVD cleavage recognition site is DTLD (Xu et al, 1996); indicating that the cleavage site is conserved between the mouse -and the rat MEKKl proteins and further supports its importance in MEKKl function.
  • ICE-like proteases are also required at a second step that is downstream of the cleavage of MEKKl because p35 inhibits the apoptosis induced by the kinase domain of MEKKl .
  • Fig. 6 shows a model defining the involvement of MEKKl in apoptosis.
  • the 196 kDa MEKKl protein can be activated by many extracellular inputs including tyrosine kinase encoded growth factor receptors, G protein-coupled receptors (Avdi et al, 1996) and cellular stresses.
  • Activation of MEKKl correlates with its phosphorylation. It is unclear at present if MEKKl phosphorylation involves autophosphorylation or additional kinases.
  • Activated MEKKl independent of its proteolysis is capable of regulating the c-Jun kinase pathway and may also regulate the ERK pathway. Both of these pathways can stimulate anti-apoptotic responses.
  • Genotoxic stress A balance between rescue and suicide using MEKKl as a switch.
  • MEKKl can function as a switch point, regulated by a proteolytic event controlled by ICE/FLICE proteases, that determines cell fate in response to a stress stimulus. Before cleavage MEKKl induces rescue mechanisms and - 70 -
  • MEKKl triggers apoptosis.
  • the cleavage of MEKKl may thus occur when the cell has failed to successfully repair itself.
  • the cleaved MEKKl then triggers apoptosis which leads to the elimination of the cell.
  • MEKKl as a protease substrate that when activated and cleaved stimulates an apoptotic response.
  • the proteolytic cleavage of MEKKl defines the mechanism to generate a protein kinase whose activity is sufficient to induce apoptosis.
  • the finding that the activation and cleavage of MEKKl occurs in response to genotoxic agents is particularly important.
  • expression of MEKKl is capable of killing by apoptosis cells that have both p53 alleles mutated.
  • the activation and cleavage of MEKKl is an apoptotic pathway that does not require a functional p53 and stimulation of these events could enhance the killing of many different tumors.

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Abstract

La présente invention concerne des protéines MEKK1 isolées, des molécules d'acide nucléique comprenant des séquences codant ces protéines et des anticorps agissant contre ces protéines. La présente invention concerne également des méthodes d'utilisation de ces protéines pour réguler une apoptose. L'invention concerne des fragments actifs de protéines MEKK1 générés par clivage de MEKK1 avec une protéase de caspase. Ces fragments actifs sont capables de stimuler l'apoptose. En outre, l'invention concerne des formes résistantes à la protéase de protéine MEKK1 qui sont résistantes au clivage par des protéases de caspase et qui sont capables d'inhiber une apoptose. L'invention concerne enfin des méthodes de génération d'un fragment actif de MEKK1, des méthodes d'identification de modulateurs de l'activité apoptotique d'un fragment actif de MEKK1 et des méthodes d'identification de modulateurs de clivage par caspase de MEKK1.
PCT/US1999/002974 1997-02-14 1999-02-12 Proteines mekk1 et leurs fragments destines a etre utilises dans la regulation d'une apoptose WO1999041385A1 (fr)

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WO2001030833A1 (fr) * 1999-10-22 2001-05-03 Shanghai Bio Road Gene Development Ltd. Nouveau polypeptide, proteine de regulation d'un nouveau cycle cellulaire 53, et polynucleotide codant pour ce polypeptide
DE10020138A1 (de) * 2000-04-14 2001-10-31 Ulf R Rapp Nukleinsäure codierend für zumindest eine raf Teilsequenz mit einer MEKK1 Bindungsstelle
EP1200573A1 (fr) * 1999-07-23 2002-05-02 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la proteine kinase 1 activee par mitogene (mekk1)
EP1402262A2 (fr) * 2001-06-05 2004-03-31 Exelixis, Inc. Map3ks utiles en tant que modificateurs de la voie p53 et methodes d'utilisation
US8268548B2 (en) 2001-06-05 2012-09-18 Exelixis, Inc. MAP3Ks as modifiers of the p53 pathway and methods of use

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1200573A1 (fr) * 1999-07-23 2002-05-02 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la proteine kinase 1 activee par mitogene (mekk1)
EP1200573A4 (fr) * 1999-07-23 2004-09-22 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la proteine kinase 1 activee par mitogene (mekk1)
WO2001030833A1 (fr) * 1999-10-22 2001-05-03 Shanghai Bio Road Gene Development Ltd. Nouveau polypeptide, proteine de regulation d'un nouveau cycle cellulaire 53, et polynucleotide codant pour ce polypeptide
DE10020138A1 (de) * 2000-04-14 2001-10-31 Ulf R Rapp Nukleinsäure codierend für zumindest eine raf Teilsequenz mit einer MEKK1 Bindungsstelle
EP1402262A2 (fr) * 2001-06-05 2004-03-31 Exelixis, Inc. Map3ks utiles en tant que modificateurs de la voie p53 et methodes d'utilisation
EP1402262A4 (fr) * 2001-06-05 2005-05-11 Exelixis Inc Map3ks utiles en tant que modificateurs de la voie p53 et methodes d'utilisation
US8268548B2 (en) 2001-06-05 2012-09-18 Exelixis, Inc. MAP3Ks as modifiers of the p53 pathway and methods of use

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