Novel phospholipase D polypeptide and DNA sequences

Abstract

Provided are novel phospholipase D DNA and amino acid sequences. The sequences are useful in methods and compositions for identifying phospholipase D mediator molecules which are in turn useful in therapeutic pharmacuetical compositions for treating rheumatoid arthritis, psoriasis, ulcerative colitis, in wound healing and for treating other diseases or conditions characterized by exhibition of an inflammatory response or in the treatment of cancer and other diseases characterized by pathogenic mitogenicity.

What is claimed is:  
     
         1 . An isolated DNA sequence that codes on expression for a PLD polypeptide.  
     
     
         2 . An isolated DNA sequence that codes on expression for a PLD1a polypeptide.  
     
     
         3 . An isolated DNA sequence that codes on expression for a PLD1b polypeptide.  
     
     
         4 . An isolated DNA sequence that codes on expression for a PLD2 polypeptide.  
     
     
         5 . An isolated DNA sequence selected from the group consisting of: 
 a) the DNA corresponding to the nucleotide sequence of SEQ ID NO:1;    b) the DNA corresponding to the nucleotide sequence of SEQ ID NO:3;    c) the DNA corresponding to the nucleotide sequence of SEQ ID NO:4;    d) the DNA corresponding to the nucleotide sequence of SEQ ID NO:6;    e) the DNA corresponding to the nucleotide sequence of SEQ ID NO:7;    f) the DNA corresponding to the nucleotide sequence of SEQ ID NO:9; and    g) a DNA capable of hybridizing under stringent conditions, or which would be capable of hybridizing under said conditions but for the degeneracy of the genetic code, to a sequence corresponding to the sequence of SEQ ID NO:1, or SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:9.    
     
     
         6 . A vector comprising a DNA sequence of  claim 1 ,  2 ,  3 ,  4  or  5 .  
     
     
         7 . A cell transformed with a DNA sequence of  claim 1 ,  2 ,  3 ,  4  or  5 , said DNA sequence being arranged in operative association with an expression control sequence capable of directing replication and expression of said DNA sequence.  
     
     
         8 . A cell according to  claim 7  wherein said cell is a mammalian cell or an insect cell.  
     
     
         9 . A process for producing a PLD protein comprising culturing a cell of  claim 7  in a suitable culture medium and isolating said PLD protein from said cell.  
     
     
         10 . A method for identifying mediators of PLD comprising the step of: 
 a) transfecting a cell line with an expression vector comprising nucleic acid sequences encoding a PLD protein;    b) culturing said cell line in a culture medium, whereby said PLD protein is expressed stably;    c) adding an effective amount of a compound to said culture medium, sufficient to cause a detectable loss in the catalytic activity of PLD; and    d) detecting said loss in catalytic activity.    
     
     
         11 . A method of  claim 10  wherein said detection is by an immunoassay.  
     
     
         12 . A method of  claim 10  wherein said PLD is labeled.  
     
     
         13 . A pharmaceutical composition useful in the treatment of wound healing comprising a therapeutically effective amount of a PLD mediator identified using the method of  claim 10  in a pharmaceutically acceptable vehicle.  
     
     
         14 . A pharmaceutical composition useful in the treatment of autoimmune diseases comprising a therapeutically effective amount of a PLD mediator identified using the method of  claim 10  in a pharmaceutically acceptable vehicle.  
     
     
         15 . A pharmaceutical composition useful in the treatment of inflammatory diseases comprising a therapeutically effective amount of a PLD mediator identified using the method of  claim 10  in a pharmaceutically acceptable vehicle.  
     
     
         16 . A polypeptide substantially free of association with other polypeptides and comprising an amino acid sequence selected from the group consisting of 
 a) the amino acid sequence of SEQ ID NO:2;    b) the amino acid sequence of SEQ ID NO:5; and    c. the amino acid sequence of SEQ ID NO:8.    
     
     
         17 . A polypeptide encoded by a DNA sequence of  claim 5 .  
     
     
         18 . A polypeptide substantially free of association with other polypeptides and comprising an enzyme of mammalian origin having a phosphatidylcholine-specific phospholipase D activity and containing at least two copies of the amino acid motif HXKXXXXD.  
     
     
         19 . A polypeptide substantially free of association with other polypeptides and comprising a PLD polypeptide that: 
 a) is perinuclear membrane associated,    b) requires PI(4,5)P2 for in vitro activity and    c) is activated by one or more G-proteins.    
     
     
         20 . A polypeptide substantially free of association with other polypeptides and comprising a PLD polypeptide that: 
 a) is plasma membrane associated,    b) activates cytoskelatal reorganization pathways,    c) requires PI(4,5)P2 for in vitro activity and    d) does not require Rac1, cdc42, RhoA, PKC or ARF1 for activation.    
     
     
         21 . A composition comprising a PLD1 or PLD2 of polypeptide of  claim 18 ,  claim 19  or  claim 20  in combination with a G-protein.  
     
     
         22 . A composition of  claim 21  wherein said G-protein is selected from the group consisting of ADP-ribosylation factor 1, RhoA, Rac1 and cdc42.