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.
Highlights
From the ‡Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, 40126 Bologna, Italy; §Institute of Molecular Genetics, National Research Council of Italy (IGM-CNR), 40126 Bologna, Italy; ¶SC Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopedic Institute, 40126 Bologna, Italy; ʈLaboratory RAMSES, Rizzoli Orthopedic Institute, 40126 Bologna, Italy
AP-MS/MS Identification of Proteins Interacting with PLC1b in the Nucleus—murine erythroleukemia (MEL)/PI-PLC1b cells were obtained by infecting MEL cells with a retroviral vector containing the coding sequence for PI-PLC1b and selection in the presence of blasticidin
In order to determine what proteins interact with PI-PLC1b in the nucleus, three independent biological experiments were conducted in which nuclei of MEL/PI-PLC1b cells were isolated during logarithmic growth
Summary
During G1/S transition, overexpression of PI-PLC1 results in up-regulation of the cyclin D3/cdk complex This complex is responsible for the phosphorylation of retinoblastoma protein and the subsequent activation of the E2F-1 transcription factor, forcing cells out of the G1 phase of the cell cycle. In G2/M, the production of inositol-3-phosphate and diacylglycerol (DAG) from the cleavage of phosphatidylinositol-4,5-bisphosphate results in PKC␣-dependent phosphorylation of lamin B, leading to nuclear envelope disassembly and cell cycle progression. The regulation of these events falls to the activation of JNK, which translocates to the nucleus and mediates PI-PLC1 phosphorylation and activation and DAG production. Nuclear PI-PLC1-induced signaling constitutes an autonomous lipid-dependent signaling system independent dysplastic syndrome; MEL, murine erythroleukemia; PI-PLC1, phosphoinositide-dependent phospholipase C beta 1
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