Phosphoinositide-specific Phospholipase C β 1b (PI-PLCβ1b) Interactome: Affinity Purification-Mass Spectrometry Analysis of PI-PLCβ1b with Nuclear Protein

Phosphoinositide-dependent phospholipase C β 1 isoform b (PI-PLCβ1b)1 is one of two existing isoforms of PI-PLCβ1 produced by alternative splicing (1). PI-PLCβ1a (150 kDa) and PI-PLCβ1b (140 kDa) differ only at their carboxyl termini; PI-PLCβ1b is 43 amino acids shorter than PI-PLCβ1a. 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-PLCβ1a, PI-PLCβ1b is almost exclusively nuclear. Nuclear PI-PLCβ1 is known to be involved in specific signal transduction pathways that differ from those occurring in other cellular compartments. The role of PI-PLCβ1 has been extensively studied in the nucleus, and PI-PLCβ1 is now considered a key co-factor for several nuclear processes, including cell growth, proliferation, and differentiation. In MEL cells, PI-PLCβ1 is involved in regulating G1/S (3) and G2/M (4) cell cycle progression. During G1/S transition, overexpression of PI-PLCβ1 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 PKCα-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-PLCβ1 phosphorylation and activation and DAG production. PI-PLCβ1 is also implicated in hematopoietic (5–8), skeletal muscle (9, 10), and adipocyte (11) differentiation. In particular, PI-PLCβ1 has different effects depending on the cell type, promoting the differentiation of myoblasts to myotubes (12) and of pre-adipocytes to adipocytes (11) but inhibiting the in vitro erythroid differentiation of both MEL cells and human CD34+ cells (5, 8). Indeed, nuclear PI-PLCβ1-induced signaling constitutes an autonomous lipid-dependent signaling system independent from its plasma membrane counterpart. The understanding of the nuclear protein network behind PI-PLCβ1 function might give insight as to the downstream target effectors and further clarify its nuclear signaling cascade. To date, our group has identified few protein targets that physically associate with PI-PLCβ1 in the nucleus, but a systematic analysis of the PI-PLCβ1 protein interactome has not been undertaken previously. By means of immune-affinity purification–mass spectrometry analysis (AP-MS), we identified 160 proteins in complex with PI-PLCβ1b in the nucleus of MEL cells. Among these, we found two interactors already known to associate with PI-PLCβ1, Srsf3 and Lmnb1.