Cytokine-, stress-, and oncoprotein-activated human protein kinase kinases

Abstract

Disclosed are human mitogen-activated (MAP) kinase kinase isoforms (MKKs). MKKs mediate unique signal transduction pathways that activate human MAP kinases p38 and JNK, which result in activation of other factors, including activating transcription factor-2 (ATF2) and c-Jun. The pathways are activated by a number of factors, including cytokines and environmental stress. Methods are provided for identifying reagents that modulate MKK function or activity and for the use of such reagents in the treatment of MKK-mediated disorders.

1 . A substantially pure human mitogen-activated protein kinase kinase (MKK) polypeptide having serine, threonine, and tyrosine kinase activity, and phosphorylating human mitogen-activated protein (MAP) kinase p38.  
     
     
         2 . A polypeptide of  claim 1  comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         3 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 2 .  
     
     
         4 . An isolated and purified polynucleotide sequence of  claim 3  consisting of the sequence of SEQ ID NO:1 or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO:1 or degenerate variants thereof.  
     
     
         5 . An isolated and purified polynucleotide sequence of  claim 3  consisting of a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO:1.  
     
     
         6 . A polypeptide of  claim 1  comprising the amino acid sequence of SEQ ID NO:4.  
     
     
         7 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 6 .  
     
     
         8 . An isolated and purified polynucleotide sequence of  claim 3  consisting of the sequence of SEQ ID NO:3 or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO:3 or degenerate variants thereof.  
     
     
         9 . An isolated and purified polynucleotide sequence of  claim 7  consisting of a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO:3.  
     
     
         10 . A polypeptide of  claim 1 , further characterized in that said polypeptide phosphorylates human mitogen-activated protein (MAP) kinase JNK.  
     
     
         11 . A polypeptide of  claim 10  comprising the amino acid sequence of SEQ ID NO:6.  
     
     
         12 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 11 .  
     
     
         13 . An isolated and purified polynucleotide sequence of  claim 12  consisting of the sequence of SEQ ID NO:5 or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO:5 or degenerate variants thereof.  
     
     
         14 . An isolated and purified polynucleotide sequence of  claim 12  consisting of a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO:5.  
     
     
         15 . A polypeptide of  claim 10  comprising an amino acid sequence of SEQ ID NO:8.  
     
     
         16 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 15 .  
     
     
         17 . An isolated and purified polynucleotide sequence of  claim 16  consisting of the sequence of SEQ ID NO:7 or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO:7 or degenerate variants thereof.  
     
     
         18 . An isolated and purified polynucleotide sequence of  claim 16  consisting of a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO:7.  
     
     
         19 . A polypeptide of  claim 10  comprising the amino acid sequence of SEQ ID NO:10.  
     
     
         20 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 19 .  
     
     
         21 . An isolated and purified polynucleotide sequence of  claim 20  consisting of the sequence of SEQ ID NO:9 or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO:9 or degenerate variants thereof.  
     
     
         22 . A recombinant expression vector comprising a polynucleotide sequence of any one of claims  3 ,  7 ,  12 ,  16 , or  20 .  
     
     
         23 . A recombinant host cell comprising a polynucleotide sequence of any one of claims  3 ,  7 ,  12 ,  16 , or  20 .  
     
     
         24 . A purified antibody which binds specifically to a polypeptide of any one of claims  1 ,  2 ,  6 ,  10 ,  11 ,  15 , or  19 .  
     
     
         25 . A method of measuring the activity of a mitogen-activated protein kinase kinase (MKK) in a biological test sample, said method comprising: 
 a) incubating said test sample with an MKK substrate for the MKK polypeptide of  claim 1  and labeled phosphate under conditions sufficient to allow phosphorylation of said substrate, and    b) determining the rate of incorporation of labeled phosphate into said substrate, wherein said rate of incorporation is a measure of MKK activity.    
     
     
         26 . A method of  claim 25 , wherein said MKK substrate is selected from the group consisting of p38 and JNK MAP kinases, activating transcription factor-2 (ATF2), ATFa, cAMP response element binding protein (CRE-BPa), and c-Jun.  
     
     
         27 . A method of  claim 25 , wherein said biological test sample is fluid, cells, or tissue obtained from a mammal.  
     
     
         28 . A method for measuring the synthesis of MKK in a biological test sample, comprising the steps of: 
 a) fractionating proteins present in said sample by gel electrophoresis;    b) transferring the proteins onto a membrane; and    c) probing the proteins with a labeled antibody specific to a MKK polypeptide of  claim 1 , wherein the level of MKK synthesis is determined by the amount of bound labeled antibody.    
     
     
         29 . A method for measuring the level of expression of MKK in a test sample, comprising the steps of: 
 a) isolating polyadenylated RNA from the test sample;    b) incubating polyadenylated RNA with a polynucleotide probe specific for a MKK polypeptide of  claim 1;     c) determining the amount of said probe hybridized said polyadenylated RNA, wherein the level of expression of MKK is directly related to the amount of MKK probe hybridized to said RNA.    
     
     
         30 . A method for identifying a reagent which modulates MKK synthesis, said method comprising: 
 a) using the method of  claim 28;     b) comparing the effect of said reagent on MKK synthesis relative to a control, wherein a reagent able to modulate MKK synthesis is identified.    
     
     
         31 . A method of  claim 30  wherein said MKK substrate is one or more of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.  
     
     
         32 . A method of  claim 30  wherein said modulation is inhibition of MKK synthesis.  
     
     
         33 . A substantially pure human mitogen-activated protein kinase kinase (MKK) polypeptide of any one of claims  1 ,  2 ,  6 ,  10 ,  11 ,  15 , or  19  for use in treating an MKK-mediated disorder.  
     
     
         34 . The polypeptide of  claim 33 , wherein said MKK-mediated disorder is selected from the group consisting of ischemic heart disease, kidney failure, oxidative liver damage, respiratory distress syndrome, heat and radiation burns, septic shock, rheumatoid arthritis, autoimmune disorders, and inflammatory diseases.  
     
     
         35 . The use of a polypeptide of  claim 33  for the manufacture of a medicament for the treatment of an MKK-mediated disorder.  
     
     
         36 . A kit useful for the detection of MKK, said kit comprising a buffer and a reagent which binds to a MKK polypeptide of any one of claims  1 ,  2 ,  6 ,  10 ,  11 ,  15 , or  19 , wherein a sample to be tested is mixed with said buffer and said reagent, and wherein said reagent is labeled.  
     
     
         37 . A kit of  claim 36 , wherein said reagent is an antibody that specifically binds MKK.