A polybasic motif in ErbB3-binding protein 1 (EBP1) has key functions in nucleolar localization and polyphosphoinositide interaction.
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 EBP1 in 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 EBP1 in 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.
NucReg Research Program, Department of Molecular Biology, University of Bergen, 5008 Bergen, Norway.
Thomas
T
Karlsson
NucReg Research Program, Department of Molecular Biology, University of Bergen, 5008 Bergen, Norway.
Altanchimeg
A
Altankhuyag
NucReg Research Program, Department of Molecular Biology, University of Bergen, 5008 Bergen, Norway.
Olena
O
Dobrovolska
NucReg Research Program, Department of Molecular Biology, University of Bergen, 5008 Bergen, Norway.
Diana C
DC
Turcu
NucReg Research Program, Department of Molecular Biology, University of Bergen, 5008 Bergen, Norway aurelia.lewis@uib.no.
Aurélia E
AE
Lewis
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C101633:PA2G4 protein, human; C095938:Pa2g4 protein, mouse; D018129:Phosphatidylinositol Phosphates; D016601:RNA-Binding Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D008239:Lysine
EBP1; PI3K; PIP3; interaction; nucleolus; p110β