Two isoforms of inositide-dependent phospholipase C 1 (PI-PLC1) are generated by alternative splicing (PLC1a and PLC1b). Both isoforms are present within the nucleus, but in contrast to PLC1a, the vast majority of PLC1b is nuclear. In mouse erythroid leukemia cells, PI-PLC1 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-PLC1, 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-PLC1 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-PLC1b, 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. Molecular & Cellular Proteomics 12: 10.1074/ mcp.M113.029686, 2220-2235, 2013.1 is one of two existing isoforms of PI-PLC1produced by alternative splicing (1). PI-PLC1a (150 kDa) and PI-PLC1b (140 kDa) differ only at their carboxyl termini; PI-PLC1b is 43 amino acids shorter than PI-PLC1a. Both isoforms present with a non-canonical nuclear localization signal comprising a cluster of lysine residues (2). In murine erythroleukemia (MEL) cells, both isoforms are present within the nucleus; however, in contrast to PI-PLC1a, PI-PLC1b is almost exclusively nuclear. Nuclear PI-PLC1 is known to be involved in specific signal transduction pathways that differ from those occurring in other cellular compartments. The role of PI-PLC1 has been extensively studied in the nucleus, and PI-PLC1 is now considered a key co-factor for several nuclear processes, including cell growth, proliferation, and differentiation.In MEL cells, PI-PLC1 is involved in regulating G1/S (3) and G2/M (4) cell cycle progression. During G1/S transition, overexpression of PI-PLC1 results in up-regulation of the cyclin D3/cdk4 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 n...