Specific binding of the C-terminal Src homology 2 domain of the p85alpha subunit of phosphoinositide 3-kinase to phosphatidylinositol 3,4,5-trisphosphate. Localization and engineering of the phosphoinositide-binding motif.

Tsui-Ting Ching,Ching-Shih Chen,Marcos Oliveira,Ho-Pi Lin,Pei-Jung Lu,Chih-Cheng Yang

doi:10.1074/jbc.m105159200

Abstract

Phosphoinositide second messengers, generated from the action of phosphoinositide 3-kinase (PI3K), mediate an array of signaling pathways through the membrane recruitment and activation of downstream effector proteins. Although pleckstrin domains of many target proteins have been shown to bind phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and/or phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) with high affinity, published data concerning the phosphoinositide binding specificity of Src homology 2 (SH2) domains remain conflicting. Using three independent assays, we demonstrated that the C-terminal (CT-)SH2 domain, but not the N-terminal SH2 domain, on the PI3K p85alpha subunit displayed discriminative affinity for PIP(3). However, the binding affinity diminished precipitously when the acyl chain of PIP(3) was shortened. In addition, evidence suggests that the charge density on the phosphoinositol ring represents a key factor in determining the phosphoinositide binding specificity of the CT-SH2 domain. In light of the largely shared structural features between PIP(3) and PI(4,5)P(2), we hypothesized that the PIP(3)-binding site on the CT-SH2 domain encompassed a sequence that recognized PI(4,5)P(2). Based on a consensus PI(4,5)P(2)-binding sequence (KXXXXXKXKK; K denotes Arg, Lys, and His), we proposed the sequence (18)RNKAENLLRGKR(29) as the PIP(3)-binding site. This binding motif was verified by using a synthetic peptide and site-directed mutagenesis. More importantly, neutral substitution of flanking Arg(18) and Arg(29) resulted in a switch of ligand specificity of the CT-SH2 domain to PI(4,5)P(2) and PI(3,4)P(2), respectively. Together with computer modeling, these mutagenesis data suggest a pseudosymmetrical relationship in the recognition of the phosphoinositol head group at the binding motif.

Highlights

Phosphoinositide second messengers, generated from the action of phosphoinositide 3-kinase (PI3K), mediate an array of signaling pathways through the membrane recruitment and activation of downstream effector proteins
We demonstrated that the p85␣ CT-Src homology 2 (SH2) domain displayed clear binding specificity and affinity for PIP3, whereas the N-terminal (NT-)SH2 domain lacked significant binding to PIP3
This study is aimed at resolving a controversial issue of whether p85␣ SH2 domains could interact with PIP3 in a selective manner

Summary

Introduction

Phosphoinositide second messengers, generated from the action of phosphoinositide 3-kinase (PI3K), mediate an array of signaling pathways through the membrane recruitment and activation of downstream effector proteins. Pleckstrin domains of many target proteins have been shown to bind phosphatidylinositol 3,4,5-trisphosphate (PIP3) and/or phosphatidylinositol 3,4-bisphosphate (PI[3,4]P2) with high affinity, published data concerning the phosphoinositide binding specificity of Src homology 2 (SH2) domains remain conflicting. Substantial evidence indicates that the lipid products of phosphoinositide 3-kinase (PI3K1), phosphatidylinositol 3,4,5trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PI[3,4]P2), facilitate transmembrane signaling, in part, by serving as membrane-localization elements to recruit target proteins to specific sites [1, 2]. It has been proposed that PIP3 binds selectively to the p85 Cterminal (CT-)SH2 domain of PI3K, thereby blocking the binding of p85 to tyrosine-phosphorylated proteins [3] This model, was refuted by a recent NMR study that showed lack of specific binding of PIP3 to p85 SH2 domains [13]. In view of the pivotal role of PI3K lipid products in cell proliferation and survival, identification of their downstream effectors may help identify new therapeutic targets

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