Constitutively active phosphatidylinositol 3-kinase and uses thereof

Abstract

The invention provides a method of producing a constitutively active phosphatidylinositol 3-kinase (PI 3-kinase) comprising the catalytic p110 subunit covalently attached at the N-terminus to the iSH2 region of the regulatory subunit, p85. The invention discloses one form of the constitutively active kinase, p110*, which functions independently of growth factor stimulation. Expression vectors encoding a constitutively active PI 3-kinase and cells containing such expression vectors are provided. The invention also provides methods of using the constitutively active phosphatidylinositol 3-kinase to generate phosphoinositides, to identify cellular target proteins and associating molecules of PI 3-kinase, to screen for inhibitors of PI 3-kinase activity and to treat certain diseases, in particular, proliferative diseases. Kits comprising the constitutively active kinase are also provided.

1 . A constitutively active phosphatidylinositol 3-kinase polypeptide comprising p85 subunit iSH2 domain sequences linked at the carboxy-terminus by a linker to the amino-terminus of a p110 subunit.  
   
   
       2 . The polypeptide of  claim 1 , wherein the N-terminal 20 amino acids are eliminated from the p110 subunit.  
   
   
       3 . The polypeptide of  claim 1 , wherein the iSH2 domain sequences consist essentially of amino acids 466 to 567 of the p85 subunit.  
   
   
       4 . The polypeptide of  claim 1  further comprising a tag at the amino or carboxy terminus.  
   
   
       5 . The polypeptide of  claim 4 , wherein the tag is an epitope.  
   
   
       6 . The polypeptide of  claim 5 , wherein the epitope tag is a myc epitope at the carboxy terminus of the p110 subunit.  
   
   
       7 . A constitutively active phosphatidylinositol 3-kinase polypeptide, comprising amino acids 466 to 567 of the p85 subunit iSH2 domain linked by a 10 amino acid glycine kinker to a p110 subunit at the amino-terminus of the p110 subunit, and a myc epitope as defined by SEQ ID NO. ______ (EQKLISEEDL) fused to the carboxy terminus of the p110 subunit.  
   
   
       8 . An expression vector comprising a DNA sequence encoding a polypeptide according to  claim 1 .  
   
   
       9 . An expression vector comprising a DNA sequence encoding a polypeptide according to  claim 4 .  
   
   
       10 . The vector of  claim 9 , also comprising pGC plasmid sequences.  
   
   
       11 . An expression vector comprising a DNA sequence encoding a polypeptide according to  claim 7 , said vector being the plasmid pCG-p110•myc.  
   
   
       12 . A cell containing the expression vector of  claim 8  or  11 .  
   
   
       13 . A method of producing a constitutively active phosphatidylinositol 3-kinase polypeptide, comprising introducing the expression vector of  claim 8  or  11  into a host cell under conditions which favor expression of the polypeptide and isolating the resultant expressed polypeptide.  
   
   
       14 . The method of  claim 13 , wherein the host cell is selected from the group consisting of a smooth muscle cell, a 293 cell, and a CHO cell.  
   
   
       15 . The method of  claim 13 , wherein the host cell is a mammalian fibroblast cell.  
   
   
       16 . The method of  claim 15 , wherein the fibroblast cell is selected from the group consisting of a COS 7 cell, a NIH 3T3 cell and a rat 3YI cell.  
   
   
       17 . A method of producing an inositol phosphate product comprising reacting a phosphatidylinositol 3-kinase polypeptide of  claim 1  or  7  with a phosphoinositide lipid substrate under appropriate kinase reaction conditions, and isolating the resultant product.  
   
   
       18 . The method of  claim 17 , wherein the product is phosphatidylinositol 3′-phosphate (PI 3′-P) and the lipid substrate is phosphatidylinositol (PI).  
   
   
       19 . The method of  claim 17 , wherein the product is phosphatidylinositol 3′,4′-bisphosphate (PI 3′,4′-P 2 ) and the lipid substrate is phosphatidylinositol 4′-phosphate (PI 4′-P).  
   
   
       20 . The method of  claim 17 , wherein the product is phosphatidylinositol 3′,4′,5′-phosphate (PI 3′,4′,5′-P 3 ) and the lipid substrate is phosphatidylinositol 4′,5′-bisphosphate (PI 4′,5′-P 2 ).  
   
   
       21 . A kit for preparing an inositol phosphate product, comprising: 
 a constitutively active phosphatidylinositol 3-kinase polypeptide of  claim 1  or  7 ; one or more phosphoinositide substrates; and instructions for preparing the inositol phosphate reagent.    
   
   
       22 . The kit of  claim 21 , further comprising a buffer for reconstituting said phosphatidylinositol 3-kinase polypeptide and a reaction buffer.  
   
   
       23 . The kit of  claim 21 , wherein the inositol phosphate product is selected from the group consisting of PI 3-P, PI 3′,4′-P 2 , and PI 3′,4′,5′-P 3 .  
   
   
       24 . The kit of  claim 21 , wherein the phosphoinositide substrate is selected from the group consisting of PI, PI 4′-P, and PI 4′,5′-P 2 .  
   
   
       25 . A method of identifying a cellular target protein substrate of a phosphatidylinositol 3-kinase, said method comprising the steps of: 
 (a) providing a phosphatidylinositol 3-kinase polypeptide of  claim 1;     (b) providing a test cell lysate thereof;    (c) providing a negative control cell lysate not contacted with said phosphatidylinositol 3-kinase polypeptide;    (d) contacting said phosphatidylinositol 3-kinase polypeptide with said test cell lysate in the presence of labeled ATP under conditions which allow said phosphatidylinositol 3-kinase to phosphorylate any cellular target protein present in said cell lysate to form a phosphorylated target protein; and    (e) comparing said test cell lysate with said negative control lysate to detect said phosphorylated target protein in said test cell lysate and thereby identifying the cellular target protein substrate.    
   
   
       26 . The method of  claim 25 , wherein the ATP is [γ 32 P]ATP.  
   
   
       27 . The method of  claim 25 , wherein both cell lysates are from stimulated cells.  
   
   
       28 . The method of  claim 26 , wherein said phosphorylated target protein is detected by: 
 performing SDS-PAGE on said cell lysates after step (d) to separate proteins contained therein, on a gel matrix; and    preparing an autoradiograph of said gel to detect radiolabeled bands present in the test cell lysates, but absent from the negative control lysate, as indicative of the presence of said phosphorylated target protein in the test cell lysate.    
   
   
       29 . A method for identifying an associating protein of an active phosphatidylinositol 3-kinase, comprising: 
 expressing a constitutively active phosphatidylinositol 3-kinase polypeptide of  claim 1  or  7  in a mammalian cell under conditions which favor expression of the polypeptide;    biosynthetically labeling proteins of said active kinase expressing cell to produce labeled proteins;    obtaining a lysate from said cell;    immunoprecipitating said expressed phosphatidylinositol 3-kinase polypeptide from said cell lysate to produce an immunocomplex;    detecting a labeled protein that co-immunoprecipitates with said phosphatidylinositol 3-kinase polypeptide, within said immunocomplex, wherein said co-immunoprecipitating protein is considered an associating protein.    
   
   
       30 . The method of  claim 29 , wherein said co-immunoprecipitating protein is detected by solubilizing said immunocomplex to release labeled proteins contained therein; separating said released proteins by SDS-PAGE; and performing autoradiography to detect a labeled protein other than said phosphatidylinositol 3-kinase polypeptide, said labeled protein being an associating protein.  
   
   
       31 . A method for identifying an associating protein of an active phosphatidylinositol 3-kinase, comprising: 
 exposing a phage or bacterial peptide library to a constitutively active phosphatidylinositol 3-kinase polypeptide of  claim 1  or  7  to allow binding of a peptide from said library to said constitutively active phosphatidylinositol 3-kinase polypeptide to form a bound peptide; and    isolating said bound peptide, wherein said bound peptide is considered an associating protein.    
   
   
       32 . The method of  claim 31 , wherein said bound peptide is isolated by affinity purification.  
   
   
       33 . A method of screening for an inhibitor of phosphatidylinositol 3-kinase activity, said method comprising: 
 providing a constitutively active phosphatidylinositol 3-kinase polypeptide of  claim 1  or  7 ;    exposing one or more test compounds to said phosphatidylinositol 3-kinase polypeptide in the presence of a phosphatidylinositol 3-kinase substrate and [ 32 P]ATP to allow phosphorylation of said substrate; and    assaying for the presence of phosphorylated substrate wherein the absence of phosphorylated substrate is indicative that the test compound is an inhibitor of phosphatidylinositol 3-kinase activity.    
   
   
       34 . The method of  claim 33 , wherein said substrate is phosphatidylinositol and said phosphorylated substrate is phosphatidylinositol 3′-P.  
   
   
       35 . A method of treating a disease selected from the group consisting of proliferative, inflammatory, allergic and cardiovascular diseases, comprising administering to a patient, a therapeutic formulation comprising an inhibitor of phosphatidylinositol 3-kinase activity in an amount effective to block phosphatidylinositol 3-kinase activity in affected cells of said patient.  
   
   
       36 . The method of  claim 35 , wherein the proliferative disease is cancer or psoriasis.  
   
   
       37 . A method of promoting wound healing, comprising administering to a patient, a therapeutic formulation comprising a phosphatidylinositol 3-kinase polypeptide of  claim 1 .  
   
   
       38 . A formulation for treating a proliferative disease, comprising a therapeutically effective amount of an inhibitor of phosphatidylinositol 3-kinase.