Specificity and Promiscuity in Phosphoinositide Binding by Pleckstrin Homology Domains

doi:10.1074/jbc.273.46.30497

Specificity and Promiscuity in Phosphoinositide Binding by Pleckstrin Homology Domains*

From the ^‡Department of Biochemistry and Biophysics, and Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6089, the ^¶Department of Cell Biology & Oncology, Istituto di Richerche Farmacologiche “Mario Negri,” Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro CH, Italy, and the ^‖Department of Pharmacology, New York University Medical Center, Skirball Institute for Biomolecular Medicine, New York, New York 10016

Abstract

Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient “dot-blot” approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-δ₁and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) binding) binds specifically tod-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) (the PtdIns(3,4,5)P₃headgroup) with K _D = 27.3 nm, but bindsd-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P₃) or d-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P₃ from Ins(1,3,4,5)P₃ (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P₃. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant DK49207 (to E. Y. S.), Telethon Italy Grant 328/bi (to M. F.), an award from the McCabe Fund of the University of Pennsylvania (to M. A. L.), and Damon Runyon Scholar Award DRS-05 from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (to M. A. L.).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.
↵§ These two authors contributed equally to this study.
↵** To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, A606 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6089. Tel.: 215-898-3072; Fax: 215-573-4764; E-mail: mlemmon{at}mail.med.upenn.edu.
Abbreviations:

PH

pleckstrin homology

PtdIns(4

5)P₂, phosphatidylinositol 4,5-bisphosphate

PtdIns(3

4,5)P₂, phosphatidylinositol 3,4,5-trisphosphate

PLC

phospholipase

PI 3-K

phosphatidylinositol 3-kinase

PKB

protein kinase B

Grp1

general receptor for phosphoinositides-1

Btk

Bruton’s tyrosine kinase

βARK

β-adrenergic receptor kinase

Sos

Son-of-sevenless

GST

glutathioneS-transferase

DTT

dithiothreitol

PBS

phosphate-buffered saline

TBS

Tris-buffered saline

SUV

small unilamellar vesicle

PtdCho

phosphatidylcholine

PtdSer

phosphatidylserine

PtdIns

phosphatidylinositol

PlecN-PH

N-terminal PH domain from pleckstrin

ITC

isothermal titration calorimetry

EGFP

enhanced green fluorescent protein

DAGK-δ

diacylglycerol kinase-δ

IP4BP

inositol 1,3,4,5-tetrakisphosphate-binding protein

ARF

ADP-ribosylation factor

MOPS

4-morpholinepropanesulfonic acid

Ins(1

3,4)P₃, d-myo-inositol 1,3,4-trisphosphate

Ins(1

4,5)P₃,d-myo-inositol 1,4,5-trisphosphate

Ins(1

5,6)P₃, d-myo-inositol 1,5,6-trisphosphate

Ins(1

4,5,6)P₄,d-myoinositol 1,4,5,6-tetrakisphosphate

Ins(3

4,5,6)P₄, d-myo-inositol 3,4,5,6-tetrakisphosphate

Ins(1

3,4,5)P₄,d-myo-inositol 1,3,4,5-tetrakisphosphate

Ins(1

2,5,6)P₄, d-myo-inositol 1,2,5,6-tetrakisphosphate

Ins(1

3,4,6)P₄,myo-inositol 1,3,4,6-tetrakisphosphate

Ins(1

3,4,5,6)P₅, myo-inositol 1,3,4,5,6-pentakisphosphate

PtdIns-3-P

phosphatidylinositol 3-phosphate

PtdIns-4-P

phosphatidylinositol 4-phosphate

PtdIns(3

4)P₂, phosphatidylinositol 3,4-bisphosphate

InsP₆

myo-inositol hexaphosphate

Ins(1

2,6)P₃, d-myo-inositol 1,2,6-trisphosphate

Ins(3

5,6)P₃,d-myo-inositol 3,5,6-trisphosphate.
- Received June 5, 1998.
- Revision received September 2, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.