A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction.

Thomas Karlsson,Diana C Turcu,Aurélia E Lewis,Altanchimeg Altankhuyag,Olena Dobrovolska

doi:10.1042/bcj20160274

Abstract

Polyphosphoinositides (PPIns) are present in the nucleus where they participate in crucial nuclear processes, such as chromatin remodelling, transcription and mRNA processing. In a previous interactomics study, aimed to gain further insight into nuclear PPIns functions, we identified ErbB3 binding protein 1 (EBP1) as a potential nuclear PPIn-binding protein in a lipid pull-down screen. EBP1 is a ubiquitous and conserved protein, located in both the cytoplasm and nucleolus, and associated with cell proliferation and survival. In the present study, we show that EBP1 binds directly to several PPIns via two distinct PPIn-binding sites consisting of clusters of lysine residues and positioned at the N- and C-termini of the protein. Using interaction mutants, we show that the C-terminal PPIn-binding motif contributes the most to the localization of EBP1in the nucleolus. Importantly, a K372N point mutation, located within the C-terminal motif and found in endometrial tumours, is sufficient to alter the nucleolar targeting of EBP1. Our study reveals also the presence of the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110β and its product PtdIns(3,4,5)P3 together with EBP1in the nucleolus. Using NMR, we further demonstrate an association between EBP1 and PtdIns(3,4,5)P3 via both electrostatic and hydrophobic interactions. Taken together, these results show that EBP1 interacts directly with PPIns and associate with PtdIns(3,4,5)P3 in the nucleolus. The presence of p110β and PtdIns(3,4,5)P3 in the nucleolus indicates their potential role in regulating nucleolar processes, at least via EBP1.

Highlights

Phospholipids are well known to play fundamental roles as structural components of membranes and in signal transduction pathways initiated at the plasma membrane
Adding 400 mM NaCl nearly abolished all interaction detected except for PtdIns4P and PtdIns(3,5)P2. These results suggest that the interactions with PtdIns3P, PtdIns5P and with PtdIns4P and PtdIns(3,5)P2 have the highest affinity for the full length (FL) ErbB3 binding protein 1 (EBP1) protein
We have previously reported that short motifs consisting of basic residues or polybasic regions (PBR) following the sequence motif K/R-(Xn = 3-7)-K-X-K/R-K/R were implicated in PPIn-binding of nuclear proteins, suggesting a mode of interaction for PPIninteracting proteins localized in the nucleus [24]

Summary

Introduction

Phospholipids are well known to play fundamental roles as structural components of membranes and in signal transduction pathways initiated at the plasma membrane They have emerged as essential components of the nucleus in the nuclear envelope and within nuclei, in the nuclear matrix and in association with the chromatin [1,2]. PPIns regulate nuclear processes, such as protein–chromatin association, transcription, pre mRNA processing, splicing and export as well as cell cycle progression [21,22,23,24,25], by interacting with proteins containing PPIn-binding domains [26] or polybasic regions (PBR)/K/R motifs [24,27]. Other data correlate changes in the levels of nuclear PPIns to cell cycle progression [12,44,45] or apoptosis via an interaction between nucleophosmin (NPM) and PtdIns(3,4,5)P3 [46]

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Results

Conclusion