Method and product for regulating cell responsiveness to external signals

Abstract

The present invention relates to isolated MEKK proteins, nucleic acid molecules having sequences that encode such proteins, and antibodies raised against such proteins. The present invention also includes methods to use such proteins to regulate signal transduction in a cell. The present invention also includes therapeutic compositions comprising such proteins or nucleic acid molecules that encode such proteins and their use to treat animals having medical disorders including cancer, inflammation, neurological disorders, autoimmune diseases, allergic reactions, and hormone-related diseases. When MEKK is expressed, it phosphorylates and activates MKKs1-4 (also referred to as MEK-1, MEK-2 and JNKK1 and JNKK2).

1 . An isolated or recombinant MEK Kinase polypeptide comprising a MEK Kinase amino acid sequence depicted in one or more of SEQ ID Nos: 6, 8, 10, 12, or 14. 
     
     
         2 . A nucleic acid encoding a mammalian MEKK protein 
     
     
         3 . The MEKK nucleic acid of  claim 2 , which nucleic acid comprises a coding sequence represented in one of SEQ ID Nos:5, 7, 9, 11, or 13. 
     
     
         4 . The nucleic acid of  claim 2 , which nucleic acid encodes a MEKK polypeptide designated by one of SEQ ID Nos: 6, 8, 10, 12, or 14. 
     
     
         5 . A nucleic acid homolog which hybridizes under stringent conditions with one or more of SEQ ID Nos: 5, 7, 9, 11, or 13. 
     
     
         6 . A nucleic acid comprising a nucleotide sequence at least 50% percent homologous with one of SEQ ID Nos:5, 7, 9, 11, or 13, or complementary thereto. 
     
     
         7 . An nucleic acid which encodes a MEKK polypeptide, wherein said polypeptide (i) phosphorylates a MAP kinase kinase protein and (ii) binds to a ras superfamily protein. 
     
     
         8 . The nucleic acid of  claim 6 , wherein said nucleic acid encodes a polypeptide which comprises a MKK consensus binding site. 
     
     
         9 . The nucleic acid of  claim 6 , wherein said MAP kinase kinase is selected from the group consisting of p42, p44, ERK1, ERK2, JNK1, JNK2, or p38 SAPK. 
     
     
         10 . The nucleic acid of  claim 6 , wherein said nucleic acid encodes a polypeptide which comprises a rac/cdc42 binding site. 
     
     
         11 . The nucleic acid of  claim 6 , wherein said nucleic acid encodes a polypeptide which does not contain an SH2 or SH3 domain. 
     
     
         12 . The nucleic acid of  claim 6 , wherein said nucleic acid encodes a polypeptide which comprises a proline rich SH3 binding motif. 
     
     
         13 . The nucleic acid of  claim 6 , wherein said nucleic acid encodes a polypeptide which comprises a Pleckstrin Homology Domain. 
     
     
         14 . The nucleic acid of  claim 6 , wherein said nucleic acid encodes a polypeptide which is capable of regulating apoptosis in a cell. 
     
     
         15 . A nucleic acid which encodes a polypeptide which is capable of competitively inhibiting the activity of a MEKK designated in one or more of SEQ ID Nos: 6, 8, 10, 12, or 14. 
     
     
         16 . The nucleic acid of  claim 14 , wherein said nucleic acid encodes a polypeptide which is at least 50% homologous with one of SEQ ID Nos: 6, 8, 10, 12, or 14. 
     
     
         17 . An nucleic acid homolog encoding a truncated MEKK polypeptide. 
     
     
         18 . The nucleic acid of  claim 17 , wherein said polypeptide comprises a region having at least 50% homolog with the protein kinase catalytic domain represented by amino acids 361-620 of SEQ ID No:6, 361-620 of SEQ ID No:8, 366-626 of SEQ ID No:10, 631-890 or SEQ ID No:12, or 1338-1597 of SEQ ID No:14. 
     
     
         19 . The nucleic acid of  claim 17 , wherein said polypeptide comprises a region having at least 50% homolog with the serine/threonine rich regulatory domain represented by amino acids 1-360 of SEQ ID No:6, 1-360 of SEQ ID No:8, 1-365 of SEQ ID No:10, 1-630 or SEQ ID No:12, or 1-1337 of SEQ ID No:14. 
     
     
         20 . An isolated nucleic acid which encodes a protein kinase catalytic domain represented in one of SEQ ID Nos: 6, 8, 10, 12, or 14. 
     
     
         21 . The nucleic acid of  claim 20 , wherein said protein kinase catalytic domain comprises amino acids 361-620 of SEQ ID No:6, 361-620 of SEQ ID No:8, 366-626 of SEQ ID No:10, 631-890 or SEQ ID No:12, or 1338-1597 of SEQ ID No: 14. 
     
     
         22 . An isolated nucleic acid which encodes an NH2 regulatory domain represented in one of SEQ ID Nos: 6, 8, 10, 12, or 14. 
     
     
         23 . The nucleic acid of  claim 22 , wherein said NH 2  regulatory domain comprises amino acids 1-360 of SEQ ID No:6, 1-360 of SEQ ID No:8, 1-365 of SEQ ID No:10, 1-630 or SEQ ID No:12, or 1-1337 of SEQ ID No:14. 
     
     
         24 . The nucleic acid of  claim 15 , wherein said polypeptide is a fusion protein further comprising, in addition to said MEKK polypeptide, a second polypeptide sequence having an amino acid sequence unrelated to said MEKK nucleic acid sequence. 
     
     
         25 . The nucleic acid of  claim 24 , wherein said fusion protein includes, as a second polypeptide sequence, a polypeptide which functions as a detectable label for detecting the presence of said fusion protein or as a matrix-binding domain for immobilizing said fusion protein. 
     
     
         26 . The nucleic acid of  claim 15 , which nucleic acid hybridizes under stringent conditions to a nucleic acid probe having a sequence represented by at least 60 consecutive nucleotides of sense or antisense of one or more of SEQ ID Nos:5, 7, 9, 11, or 13. 
     
     
         27 . The nucleic acid of  claim 15 , further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector. 
     
     
         28 . An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of  claim 15 . 
     
     
         29 . A host cell transfected with the expression vector of  claim 28  and expressing said recombinant polypeptide. 
     
     
         30 . A method of producing a recombinant MEKK polypeptide comprising culturing the cell of  claim 29  in a cell culture medium to express said recombinant polypeptide and isolating said recombinant polypeptide from said cell culture. 
     
     
         31 . A transgenic animal having cells which harbor a transgene encoding a MEKK polypeptide, which animals are vertebrates. 
     
     
         32 . A transgenic animal having cells in which a gene for a MEKK is disrupted, which animals are vertebrates. 
     
     
         33 . A recombinant transfection system, comprising:
 (i) a gene construct including the nucleic acid of  claim 6  and operably linked to a transcriptional regulatory sequence for causing expression of said MEKK polypeptide in eukaryotic cells, and   (ii) a gene delivery composition for delivering said gene construct to a cell and causing the cell to be transfected with said gene construct.   
     
     
         34 . The recombinant transfection system of  claim 33 , wherein the gene delivery composition is selected from a group consisting of a recombinant viral particle, a liposome, and a poly-cationic nucleic acid binding agent. 
     
     
         35 . A nucleic acid composition comprising a substantially purified oligonucleotide, said oligonucleotide including a region of nucleotide sequence which hybridizes under stringent conditions to at least 25 consecutive nucleotides of sense or antisense sequence of a vertebrate MEKK gene. 
     
     
         36 . The nucleic acid composition of  claim 35 , which oligonucleotide hybridizes under stringent conditions to at least 50 consecutive nucleotides of sense or antisense sequence of a vertebrate MEKK gene. 
     
     
         37 . The nucleic acid composition of  claim 36 , wherein said oligonucleotide further comprises a label group attached thereto and able to be detected. 
     
     
         38 . The nucleic acid composition of  claim 37 , wherein said oligonucleotide has at least one non-hydrolyzable bond between two adjacent nucleotide subunits. 
     
     
         39 . A test kit for detecting cells which contain a MEKK mRNA transcript, comprising the nucleic acid composition of  claim 37  for measuring, in a sample of cells, a level of nucleic acid encoding a MEKK protein. 
     
     
         40 . A method for modulating one or more of growth, differentiation, or survival of a mammalian cell said cell possessing or engineered to posses MEKK substrates, comprising treating the cell with an effective amount of an agent which activates or inactivates MEKK polypeptide thereby altering, relative to the cell in the absence of the agent, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell.