PI 3-kinase fusion mutants and uses thereof

Abstract

Polynucleotide constructs encoding growth factor independent catalytically active membrane targeted PI 3-kinase mutants useful for therapeutic and research purposes are described. In addition, a method for using the polynucleotide constructs to screen for inhibitors of PI 3-kinase, a method for making 3′ phosphorylated inositol phospholipids, methods of reducing cell death after trauma, and methods of overcoming insulin resistance are described.

What is claimed is:  
     
         1 . A polynucleotide sequence comprising: 
 (a) a first nucleotide sequence comprising a sequence selected from the group consisting of: 
 (i) a sequence encoding the p110 subunit of PI 3-kinase protein, and  
 (ii) a sequence encoding a derivative or mutant of (i) having a single or multiple nucleotide substitution, deletion or addition, said derivative or mutant having an activity of the p110 subunit of PI 3-kinase protein, and  
   (b) a second nucleotide sequence comprising a sequence encoding a cell membrane targeting sequence, said second nucleotide sequence being attached to the 5′ or 3′ end of said first nucleotide sequence.    
     
     
         2 . A polynucleotide sequence of  claim 1 , wherein said first nucleotide sequence further comprises an additional sequence selected from the group consisting of: 
 (i) a sequence encoding a portion of the p85 subunit of PI 3-kinase protein that is capable of binding the p110 subunit of PI 3-kinase protein, and    (ii) a sequence encoding a derivative or mutant. of (i) having a single or multiple nucleotide substitution, deletion or addition, said derivative or mutant being capable of binding the p110 subunit of PI 3-kinase.    
     
     
         3 . A polynucleotide sequence of  claim 2 , wherein said additional sequence comprises the iSH2 domain of the p85 subunit of PI 3-kinase protein.  
     
     
         4 . A polynucleotide sequence of  claim 1  wherein said cell membrane targeting sequence is selected from the group consisting of 
 (a) a myristoylation cell membrane targeting sequence, and  
 (b) farnesylation and palmitoylation cell membrane targeting sequences.  
 
     
     
         5 . A sequence of  claim 3 , wherein said first nucleotide sequence comprises a sequence encoding p110* and said second nucleotide sequence comprises a sequence encoding a cell membrane targeting sequence selected from the group consisting of: 
 (a) a myristoylation sequence and    (b) farnesylation and palmitoylation sequences.    
     
     
         6 . A polynucleotide sequence comprising: 
 (a) a first nucleotide sequence comprising a sequence selected from the group consisting of: 
 (i) a sequence encoding the p110 subunit of PI3 kinase protein, and  
 (ii) a sequence encoding a derivative or mutant of (i) having single or multiple nucleotide substitutions, deletions or additions, said derivative or mutant having an activity of the p110 subunit PI 3-kinase,  
   (b) a second nucleotide sequence comprising a sequence selected from the group consisting of: 
 (i) a sequence encoding the iSH2 domain of the p85 subunit of PI3 kinase protein that is capable of binding the p110 subunit of PI 3-kinase protein, and  
 (ii) a sequence encoding a derivative or mutant of (i) having a single or multiple nucleotide substitution, deletion or addition, said derivative or mutant being capable of binding the p110 subunit of PI 3-kinase protein, wherein said second nucleotide sequence is attached to a linker nucleotide sequence encoding a linker, said linker nucleotide sequence being attached to the 5′ end of said first nucleotide sequence and forming a first fusion sequence, and  
   (c) a third nucleotide sequence encoding a cell membrane targeting sequence, attached to the 5′ or 3′ end of said first fusion sequence.    
     
     
         7 . A polynucleotide sequence of  claim 6  wherein said cell membrane targeting sequence comprises a sequence selected from the group consisting of: 
 (a) a myristoylation cell membrane targeting sequence and  
 (b) farnesylation and palmitoylation cell membrane targeting sequences.  
 
     
     
         8 . A cell transformed with said polynucleotide sequence of  claim 1 .  
     
     
         9 . A cell transformed with said polynucleotide sequence of  claim 6 .  
     
     
         10 . A transgenic fly comprising a transgene having a polynucleotide sequence of  claim 6  under regulatory control of an eye specific promoter, wherein said fly exhibits a phenotypic change in eye morphology from normal to rough eye morphology.  
     
     
         11 . A method of screening for an inhibitor of PI 3-kinase comprising: 
 (a) administering a candidate inhibitor to a transgenic fly of  claim 10 ,    (b) observing any reversion in phenotype to normal eye morphology in said fly, said reversion being indicative of PI 3-kinase inhibitor activity.    
     
     
         12 . A method of reducing cell death due to trauma, comprising administering to a mammalian patient a viral or non-viral vector comprising a polynucleotide sequence of  claim 1 .  
     
     
         13 . A method of reducing cell death due to trauma, comprising administering to a mammalian patient a viral or non-viral vector comprising a polynucleotide sequence of  claim 6 .  
     
     
         14 . A method of making a 3′ phosphorylated inositol phospholipid comprising: 
 (a) contacting a purified p110 or p110* polypeptide with a vesicle including a PI 3kinase substrate selected from the group consisting of phosphatidylinositol (PI), phosphatidyl 4phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate (PI4,5,P 2 ), and  
 (b) isolating a 3′ phosphorylated inositol phospholipid.  
 
     
     
         15 . A method of making a 3′ phosphorylated inositol phospholipid comprising transforming a host cell with said polynucleotide of  claim 1  and expressing said polynucleotide.  
     
     
         16 . A method of making a 3′ phosphorylated inositol phospholipid comprising transforming a host cell with said polynucleotide of  claim 6  and expressing said polynucleotide.  
     
     
         17 . A 3′ phosphorylated inositol phospholipid made by the method of  claim 14 .  
     
     
         18 . A 3′ phosphorylated inositol phospholipid made by the method of  claim 16 .  
     
     
         19 . A method of activating an enzyme effector of PI 3-kinase having a pleckstrin homology domain comprising: 
 (a) incubating a polynucleotide sequence of  claim 1  with a 4′ phosphorylated phosphatidylinositol selected from the group consisting of phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate (PI4,5P 2, ) to generate a mixture of 3′ phosphorylated inositol phospholipids comprising phosphatidylinositol 3,4 bisphosphate (PI3,4P 2, ), and phosphatidylinositol 3,4,5 trisphosphate (PI3,4,5P 3, ),    (b) isolating a 3′ phosphorylated inositol phospholipid of (a) and    (c) contacting an active polypeptide having a pleckstrin homology domain with an effective amount of said isolated 3′ phosphorylated inositol phospholipid of (b).    
     
     
         20 . A method of promoting activation in a mammalian patient of an insulin signaling pathway comprising contacting a cell characterized by insulin resistance with a vector comprising a polynucleotide sequence of  claim 6 .  
     
     
         21 . A method of reducing cell death associated with trauma in a mammalian patient, comprising contacting a population of said patient's cells with an effective amount of a pharmaceutical composition comprising a 3′ phosphorylated inositol phospholipid of  claim 18.