title: Phosphoinositide-specific phospholipase C β 1b (PI-PLCβ1b) interactome: affinity purification-mass spectrometry analysis of PI-PLCβ1b with nuclear protein.
abstract: Two isoforms of inositide-dependent phospholipase C β1 (PI-PLCβ1) are generated by alternative splicing (PLCβ1a and PLCβ1b). Both isoforms are present within the nucleus, but in contrast to PLCβ1a, the vast majority of PLCβ1b is nuclear. In mouse erythroid leukemia cells, PI-PLCβ1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCβ1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCβ1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCβ1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule.
authors.affiliation: Cell Signaling Laboratory, Department of Biomedical Science DIBINEM, University of Bologna, 40126 Bologna, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Piazzi
authors.forename: William L
authors.initials: WL
authors.lastname: Blalock
authors.forename: Alberto
authors.initials: A
authors.lastname: Bavelloni
authors.forename: Irene
authors.initials: I
authors.lastname: Faenza
authors.forename: Antonietta
authors.initials: A
authors.lastname: D'Angelo
authors.forename: Nadir M
authors.initials: NM
authors.lastname: Maraldi
authors.forename: Lucio
authors.initials: L
authors.lastname: Cocco
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D054799:Phospholipase C beta / Q000378:metabolism*; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002843:Chromatin; D009687:Nuclear Proteins; D020033:Protein Isoforms; D054799:Phospholipase C beta