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J Biol Chem, Vol. 274, Issue 53, 37893-37900, December 31, 1999

Expression Cloning of Protein Targets for 3-Phosphorylated Phosphoinositides*

Vikram R. RaoDagger §, Michael N. CorradettiDagger , Jian Chen, Jirong Peng, Junying YuanDagger , Glenn D. Prestwich, and Joan S. BruggeDagger ∥

From the Dagger  Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and the  Department of Medicinal Chemistry University of Utah, Salt Lake City, Utah 84112

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 vitro transcription/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-P3 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 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 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.

* This work was supported by Grants CA27951 and CA78773 from the NCI, National Institutes of Health (to J. S. B.) and by Grants NS29632 and GM57705 (to G. D. P.) and AG12859 (to J. Y.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Special Fellowship from the Leukemia Society of America.

∥ To whom correspondence should be addressed: Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-3974; Fax: 617-432-3969; E-mail: joan_brugge@hms.harvard.edu.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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