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.

What is claimed is:  
     
         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 recombinant expression vector containing a polynucleotide sequence of  claim 3 .  
     
     
         7 . A recombinant host cell comprising a polynucleotide sequence of  claim 3 .  
     
     
         8 . A polypeptide of  claim 1  comprising the amino acid sequence of SEQ ID NO:4.  
     
     
         9 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 8 .  
     
     
         10 . 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.  
     
     
         11 . An isolated and purified polynucleotide sequence of  claim 9  consisting of a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO:3.  
     
     
         12 . A recombinant expression vector containing a polynucleotide sequence of  claim 9 .  
     
     
         13 . A recombinant host cell comprising a polynucleotide sequence of  claim 9 .  
     
     
         14 . A polypeptide of  claim 1 , further characterized in that said polypeptide phosphorylates human mitogen-activated protein (MAP) kinase JNK.  
     
     
         15 . A polypeptide of  claim 14  comprising the amino acid sequence of SEQ ID NO:6.  
     
     
         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:5 or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO:5 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:5.  
     
     
         19 . A recombinant expression vector containing a polynucleotide sequence of  claim 16 .  
     
     
         20 . A recombinant host cell comprising a polynucleotide sequence of  claim 16 .  
     
     
         21 . A polypeptide of  claim 14  comprising an amino acid sequence of SEQ ID NO:8.  
     
     
         22 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 21 .  
     
     
         23 . An isolated and purified polynucleotide sequence of  claim 22  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.  
     
     
         24 . An isolated and purified polynucleotide sequence of  claim 22  consisting of a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO:7.  
     
     
         25 . A recombinant expression vector containing a polynucleotide sequence of  claim 22 .  
     
     
         26 . A recombinant host cell containing a polynucleotide sequence of  claim 22 .  
     
     
         27 . A polypeptide of  claim 14  comprising the amino acid sequence of SEQ ID NO:10.  
     
     
         28 . An isolated and purified polynucleotide sequence encoding a polypeptide of  claim 27 .  
     
     
         29 . An isolated and purified polynucleotide sequence of  claim 28  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.  
     
     
         30 . A recombinant expression vector comprising a polynucleotide sequence of  claim 28 .  
     
     
         31 . A recombinant host cell comprising a polynucleotide sequence of  claim 28 .  
     
     
         32 . A purified antibody which binds specifically to a polypeptide of  claim 1 .  
     
     
         33 . A purified antibody which binds specifically to a polypeptide of  claim 2 .  
     
     
         34 . A purified antibody which binds specifically to a polypeptide of  claim 8 .  
     
     
         35 . A purified antibody which binds specifically to a polypeptide of  claim 14 .  
     
     
         36 . A purified antibody which binds specifically to a polypeptide of  claim 15 .  
     
     
         37 . A purified antibody which binds specifically to a polypeptide of  claim 21 .  
     
     
         38 . A purified antibody which binds specifically to a polypeptide of  claim 27 .  
     
     
         39 . 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.    
     
     
         40 . A method of  claim 39  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.  
     
     
         41 . A method of  claim 39  wherein said biological test sample is fluid, cells, or tissue obtained from a mammal.  
     
     
         42 . 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.    
     
     
         43 . 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.    
     
     
         44 . A method for identifying a reagent which modulates MKK activity, said method comprising: 
 a) using the method of claim  39 ;    b) comparing the effect of said reagent on MKK activity relative to a control, wherein a reagent able to modulate MKK substrate phosphorylation is identified.    
     
     
         45 . A method of  claim 44  wherein said MKK substrate is one or more of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.  
     
     
         46 . A method of  claim 44  wherein said modulation is inhibition of MKK activity.  
     
     
         47 . A method for identifying a reagent which modulates MKK synthesis, said method comprising: 
 a) using the method of claim  42 ;    b) comparing the effect of said reagent on MKK synthesis relative to a control, wherein a reagent able to modulate MKK synthesis is identified.    
     
     
         48 . A method of  claim 47  wherein said MKK substrate is one or more of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.  
     
     
         49 . A method of  claim 47  wherein said modulation is inhibition of MKK synthesis.  
     
     
         50 . A method for identifying a reagent which modulates MKK expression, said method comprising: 
 a) using the method of claim  43 ;    b) comparing the effect of said reagent on MKK expression relative to a control, wherein a reagent able to modulate MKK expression is identified.    
     
     
         51 . A method of treating an MKK-mediated disorder in a patient, comprising administering to the patient a therapeutically effective amount of a reagent that modulates MKK activity.  
     
     
         52 . The method of  claim 51 , 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.  
     
     
         53 . A kit useful for the detection of MKK, said kit comprising a buffer and a reagent which binds to a MKK polypeptide of  claim 1 , wherein said a sample to be tested is mixed with said buffer and said reagent, and wherein said reagent is labeled.  
     
     
         54 . A kit of  claim 53 , wherein said reagent is an antibody which specifically binds MKK.  
     
     
         55 . A kit of  claim 53 , wherein said reagent is a labeled cDNA oligonucleotide which specifically bind MKK mRNA.