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dc.contributor.authorRao, Vikram R.
dc.contributor.authorCorradetti, Michael N.
dc.contributor.authorChen, Jian
dc.contributor.authorPeng, Jirong
dc.contributor.authorYuan, Junying
dc.contributor.authorPrestwich, Glenn D.
dc.contributor.authorBrugge, Joan S.
dc.date.accessioned2019-10-05T16:05:58Z
dc.date.issued1999
dc.identifier.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.
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41483555*
dc.description.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.
dc.language.isoen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology
dash.licenseLAA
dc.titleExpression Cloning of Protein Targets for 3-Phosphorylated Phosphoinositides
dc.typeJournal Article
dc.description.versionVersion of Record
dc.relation.journalThe Journal of Biological Chemistry
dash.depositing.authorYuan, Junying::c7e832b918fc7c2356d6c49fe1a0b135::600
dc.date.available2019-10-05T16:05:58Z
dash.workflow.comments1Science Serial ID 105660
dc.identifier.doi10.1074/jbc.274.53.37893
dash.source.volume274;53
dash.source.page37893-37900


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