Methods and compositions for regulating cellular signaling

Abstract

The present invention related to methods and compositions for modulating cellular signaling. In particular, the present invention relates to PKK and RICK3 proteins. The present invention further relates the to use of PKK and RICK3 proteins in modulating NF-κB signaling. The present invention thus provides novel targets for drug screening and therapeutics.

We claim:  
     
         1 . An isolated and purified nucleic acid comprising a sequence encoding a protein selected from the group consisting of SEQ ID NOs: 3 and sequences that are at least 90% identical to SEQ ID NO: 3.  
     
     
         2 . The nucleic acid sequence of  claim 1 , wherein said sequence is operably linked to a heterologous promoter.  
     
     
         3 . The nucleic acid sequence of  claim 1 , wherein said sequence is contained within a vector.  
     
     
         4 . The nucleic acid sequence of  claim 3 , wherein said vector is within a host cell.  
     
     
         5 . An isolated and purified nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2 4, 5, 6, 7, 8, 9, and 10.  
     
     
         6 . The nucleic acid sequence of  claim 5 , wherein said nucleic acid sequence is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2 4, 5, 6, 7, 8, 9, and 10.  
     
     
         7 . The nucleic acid sequence of  claim 5 , wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 2 4, 5, 6, 7, 8, 9, and 10.  
     
     
         8 . The nucleic acid sequence of  claim 5 , wherein said sequence encodes a protein that binds to PKK.  
     
     
         9 . A vector comprising the nucleic acid sequence of  claim 5 .  
     
     
         10 . A host cell comprising the vector of  claim 9 .  
     
     
         11 . The host cell of  claim 10 , wherein said host cell is located in an organism selected from the group consisting of a plant and an animal.  
     
     
         12 . A protein encoded by a nucleic acid selected from the group consisting of SEQ ID NOs: 2 4, 5, 6, 7, 8, 9, and 10 and variants thereof that are at least 80% identical to SEQ ID NOs: 2 4, 5, 6, 7, 8, 9, and 10, wherein said protein has at least one activity of RICK3.  
     
     
         13 . The protein of  claim 12 , wherein said activity is binding to PKK.  
     
     
         14 . The protein of  claim 12 , wherein said activity is inhibition of PKK induced NF-κB activation.  
     
     
         15 . The protein of  claim 12 , wherein said protein is at least 90% identical to SEQ ID NO: 3.  
     
     
         16 . The protein of  claim 12 , wherein said protein is at least 95% identical to SEQ ID NO: 3.  
     
     
         17 . A method for producing variants of RICK3 comprising: 
 a) providing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 24,5,6,7,8,9,and 10;    b) mutating said nucleic acid sequence to generate a variant of RICK3; and    c) screening said variant for RICK3 activity.    
     
     
         18 . A composition comprising a nucleic acid that inhibits the binding of at least a portion of a nucleic acid selected from the group consisting of SEQ ID NOs: 2 4, 5, 6, 7, 8, 9, and 10 to their complementary sequences.  
     
     
         19 . A method for screening compounds, comprising: 
 a) providing: 
 i) a first polypeptide sequence comprising at least a portion of PKK;  
 ii) a second polypeptide sequence comprising at least a portion of a protein known to interact with PKK; and  
 iii) one or more test compounds;  
   b) combining in any order, said first polypeptide sequence comprising at least a portion of PKK, said second polypeptide sequence comprising at least a portion of a protein known to interact with PKK, and said one or more test compounds under conditions such that said first polypeptide sequence, said second polypeptide sequence, and said test compound interact; and    c) detecting the presence or absence of an interaction between said polypeptide sequence comprising at least a portion of PKK and said polypeptide sequence comprising at least a portion of a protein known to interact with PKK.    
     
     
         20 . The method of  claim 19 , wherein said first polypeptide comprises SEQ ID NO: 12.  
     
     
         21 . The method of  claim 19 , wherein said first polypeptide comprises a fragment of SEQ ID NO: 12.  
     
     
         22 . The method of  claim 19 , wherein said second polypeptide comprises PKC.  
     
     
         23 . The method of  claim 19 , wherein said second polypeptide comprises RICK3.  
     
     
         24 . The method of  claim 19 , wherein said test compound is a drug.  
     
     
         25 . A drug identified by the method of  claim 19 .  
     
     
         26 . A method for screening compounds, comprising: 
 a) providing: 
 i) a polypeptide sequence comprising at least a portion of a polypeptide selected from the group consisting of PKK and RICK3, wherein said polypeptide comprises protein kinase activity; and  
 iii) one or more test compounds; and  
   b) contacting said test compound and said polypeptide; and    c) detecting the level of kinase activity of said polypeptide.    
     
     
         27 . The method of  claim 26 , wherein said kinase activity is increased relative to the kinase activity in the absence of said test compound.  
     
     
         28 . The method of  claim 26 , wherein said kinase activity is decreased relative to the kinase activity in the absence of said test compound.  
     
     
         29 . The method of  claim 26 , wherein said polypeptide comprises SEQ ID NO: 12.  
     
     
         30 . The method of  claim 26 , wherein said polypeptide comprises a fragment of SEQ ID NO: 12.  
     
     
         31 . The method of  claim 26 , wherein said polypeptide comprises SEQ ID NO: 3.  
     
     
         32 . The method of  claim 26 , wherein said polypeptide comprises a fragment of SEQ ID NO: 3.  
     
     
         33 . The method of  claim 26 , wherein said test compound is a drug.  
     
     
         34 . The method of  claim 26 , further comprising providing a kinase substrate.