NDLI: Expression Cloning of Protein Targets for 3-Phosphorylated Phosphoinositides

Content Provider	Journal of Biological Chemistry (JBC)
Author	Rao, Vikram R. Corradetti, Michael N. Chen, Jian Peng, Jirong Yuan, Junying Prestwich, Glenn D. Brugge, Joan S.
Abstract	The 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 effectors 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-P2) and PtdIns-3,4,5-trisphosphate (PtdIns-3,4,5-P3). This screen uses synthetic biotinylated analogs of these lipids in conjunction with libraries of radiolabeled proteins that are produced by coupled in vitrotranscription/translation reactions. The feasibility of the screen was initially demonstrated using avidin-coated beads prebound to biotinylated PtdIns-3,4-P2 and PtdIns-3,4,5-P3to 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 from 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/p42IP4 and the PtdIns-3,4,5-P3-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-P3 and PtdIns-3,4-P2 than for PtdIns-4,5-bisphosphate. Transcripts of this novel gene are present in every tissue analyzed but are most prominently expressed in spleen. We have renamed this new protein PHISH for 3′-phosphoinositide-interacting Srchomology-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.
Related Links	http://www.jbc.org/content/274/53/37893.abstract
Ending Page	37900
Starting Page	37893
Page Count	8
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	53
Volume Number	274
DOI	10.1074/jbc.274.53.37893
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	1999-12-31
Access Restriction	Open
Subject Keyword	Phosphatidylinositol (PtdIns) ADP ribosylation factor (ARF) Expressed sequence tag (EST) Maltose-binding protein (MBP) Pleckstrin homology (PH) MBP-Akt PH domain (MBP-PH) 3′-phosphorylated phosphatidylinositol (3′-PPI) Phosphoinositide-dependent kinase 1 (PDK1) 3′-phosphoinositide interacting SH2-containing protein (PHISH) Phosphatidylinositol 3′-kinase (PI 3′-K) PtdIns-3-monophosphate (PtdIns-3-P) 4,5-P3, PtdIns-3,4,5-trisphosphate (PtdIns-3) 5-P2, PtdIns-4,5-bisphosphate (PtdIns-4) Src homology 2 (SH2) CELL BIOLOGY AND METABOLISM
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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