Expression cloning of protein targets for 3-phosphorylated phosphoinositides

The phosphatidylinositol 3-kinase (PI 3'-K) family of lipid kinases play a critical role in cell proliferation, survival, vesicle trafficking, motilit y, cytoskeletal rearrangements, and oncogenesis. To identify downstream eff ecters of PI 3'-K, we developed a novel screen to isolate proteins that bin d to the major products of PI 3'-K: phosphatidylinositol-3,4-bisphosphate ( PtdIns-3,4-P-2) and PtdIns-3,4,5-trisphosphate (PtdIns-3,4,S-P-3). This scr een uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitro t ranscription/translation reactions. The feasibility of the screen was initi ally demonstrated using avidin-coated beads prebound to biotinylated PtdIns -3,4-P-2 and PtdIns-3,4,5-P-3, to specifically isolate the pleckstrin homol ogy domain of the serine/threonine kinase Akt. We then demonstrated the uti lity of this technique in isolating novel 3'-phosphorylated phosphatidylino sitol (3'-PPI)-binding proteins through the preliminary screening of in. vi tro transcribed/translated cDNAs fi om a small pool expression library deri ved from mouse spleen. Three proteins were isolated that bound specifically to 3'PPIs. Two of these proteins have been previously characterized as PIP 3BP/p42(IP4) and the PtdIns-3,4,5-P-3-dependent serine/threonine kinase pho sphoinositide-dependent kinase 1. The third protein is a novel protein that contains only a Src homology 2 domain and a pleckstrin homology domain; th is protein has a higher specificity for both PtdIns-3,4,5-P-3 and PtdIns-3, 4-P-2 than for PtdIns-4,5-bisphosphate. Transcripts of this novel gene are present in every tissue analyzed hut are most prominently expressed in sple en. We have renamed this new protein PRISH for 3'-phosphoinositide-interact ing Src homology-containing protein. This report demonstrates the utility o f this technique for isolating and characterizing 3'-PPI-binding proteins a nd has broad applicability for the isolation of binding domains for other L ipid products.