Expression Cloning of Protein Targets for 3-Phosphorylated Phosphoinositides
Rao, Vikram R.
Corradetti, Michael N.
Prestwich, Glenn D.
Brugge, Joan S.
MetadataShow full item record
CitationRao, Vikram R., Michael N. Corradetti, Jian Chen, Jirong Peng, Junying Yuan, Glenn D. Prestwich, and Joan S. Brugge. 1999. “Expression Cloning of Protein Targets for 3-Phosphorylated Phosphoinositides.” Journal of Biological Chemistry 274 (53): 37893–900. https://doi.org/10.1074/jbc.274.53.37893.
AbstractThe phosphatidylinositol 3-kinase (PI 3'-K) family of lipid kinases play a critical role in cell proliferation, survival, vesicle trafficking, motility, cytoskeletal rearrangements, and oncogenesis. To identify downstream effecters of PI 3'-K, we developed a novel screen to isolate proteins that bind 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 screen uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitro transcription/translation reactions. The feasibility of the screen was initially 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 homology domain of the serine/threonine kinase Akt. We then demonstrated the utility of this technique in isolating novel 3'-phosphorylated phosphatidylinositol (3'-PPI)-binding proteins through the preliminary screening of in. vitro transcribed/translated cDNAs fi om a small pool expression library derived from mouse spleen. Three proteins were isolated that bound specifically to 3'PPIs. Two of these proteins have been previously characterized as PIP3BP/p42(IP4) and the PtdIns-3,4,5-P-3-dependent serine/threonine kinase phosphoinositide-dependent kinase 1. The third protein is a novel protein that contains only a Src homology 2 domain and a pleckstrin homology domain; this 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 spleen. We have renamed this new protein PRISH for 3'-phosphoinositide-interacting Src homology-containing protein. This report demonstrates the utility of this technique for isolating and characterizing 3'-PPI-binding proteins and has broad applicability for the isolation of binding domains for other Lipid products.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41483555
- HMS Scholarly Articles