Two isoforms of inositide-dependent phospholipase C beta1 (PI-PLCbeta1) are generated by alternative splicing (PLCbeta1a and PLCbeta1b). Both isoforms are present within the nucleus, but in contrast to PLCbeta1a, the vast majority of PLCbeta1b is nuclear. In mouse erythroid leukemia cells, PI-PLCbeta1 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-PLCbeta1, 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-PLCbeta1 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-PLCbeta1b, 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.
Phosphoinositide-specific phospholipase C beta1b (PI-PLCbeta1b) interactome: affinity purification-mass spectrometry analysis of PI-PLCbeta1b with nuclear protein
Piazzi M;
2013
Abstract
Two isoforms of inositide-dependent phospholipase C beta1 (PI-PLCbeta1) are generated by alternative splicing (PLCbeta1a and PLCbeta1b). Both isoforms are present within the nucleus, but in contrast to PLCbeta1a, the vast majority of PLCbeta1b is nuclear. In mouse erythroid leukemia cells, PI-PLCbeta1 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-PLCbeta1, 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-PLCbeta1 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-PLCbeta1b, 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.