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J Biol Chem, Vol. 274, Issue 53, 37893-37900, December 31, 1999
From the 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.
Expression Cloning of Protein Targets for 3-Phosphorylated
Phosphoinositides*
§,,,
Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115 and the ¶ Department of
Medicinal Chemistry University of Utah,
Salt Lake City, Utah 84112
*
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.
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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