Bavelloni Alberto, Faenza Irene, Cioffi Gabriella, Piazzi Manuela, Parisi Daniela, Matic Ivan, Maraldi Nadir M, Cocco Lucio
Laboratory of Cell Biology and Electron Microscopy, IOR, Bologna Italy.
Proteomics. 2006 Nov;6(21):5725-34. doi: 10.1002/pmic.200600318.
An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).
大量证据表明肌醇特异性磷脂酶C（PLC）与细胞核相关，位于细胞核中的主要同工型是PLCβ(1)。先前已表明，细胞核PLCβ(1)的组成型过表达会抑制弗瑞德红白血病细胞分化，并通过靶向细胞周期蛋白D3诱导细胞周期进程。鉴于PLCβ(1)信号在细胞生长和分化过程中在细胞核中所发挥的作用，本研究的目的是鉴定受PLCβ(1)过表达调控的新蛋白质。为了鉴定弗瑞德红白血病细胞中细胞核PLCβ(1)依赖性信号传导的新型下游效应物，我们进行了基于高分辨率二维电泳的蛋白质组学分析。通过蛋白质组学方法，我们发现，与野生型细胞相比，在过表达细胞核PLCβ(1)的细胞中，SRp20（一种高度保守的剪接调节因子SR家族的成员）表达下调。二维蛋白质印迹法证实了SRp20的减少。此外，我们还表明细胞核PLCβ(1)与SRp20剪接因子结合。实际上，通过免疫沉淀和亚细胞分级分离，我们证明了内源性PLCβ(1)和SRp20在细胞核中发生物理相互作用。在此我们展示了PLCβ(1)特异性靶标——剪接因子SRp20的存在，其表达受到PLCβ(1)诱发的细胞核信号传导的特异性下调。
