Studio di espressione e funzione della PLC?1 nucleare in modelli murini ed umani di cellule ematopoietiche mieloidi e linfoidi

The aims of this work were to investigate the role of nuclear Phospholipase C beta 1 (PI-PLC?1) in human and mouse cell lines and to identify new binding partners of nuclear PI-PLC?1 to further understand the functional network in which the enzyme acts. The intracellular distribution of PI-PLC?1 was further investigated in human leukaemia cell lines (NB4, HL60, THP1, CEM, Jurkat, K562). With the exception of HL60, a high endogenous level of PI-PLC?1 was detected in purified nuclei in each of the cell lines. We found that also in Ba/F3 pro-B cells overexpressing PI-PLC?1b the protein localize within the nucleus. Although our data demonstrated that PI-PLC?1b was not involved in cell proliferation and IGF-1 response as shown in other cell lines (FELC and Swiss 3T3), there was an effect on apoptosis. Activation of early apoptotic markers caspase-3 and PARP was delayed in PI-PLC?1b overexpressing Ba/F3 cells treated with 5 gr/ml mitomycin C for 24h. We performed an antibody-specific immunoprecipitation on nuclear lysates from FELC-PLC?1b cells. Mass spectrometry analysis (nano-ESI-Q-TOF) of co-immunoprecipitated proteins allowed for identification of 92 potential nuclear PI-PLC?1b interactors. Among these, several already documented PI-PLC?1b interacting partners (Srp20, LaminB, EF1?2) were identified, further validating our data. All the identified proteins were nuclear, mostly localized within the nuclear speckles. This evidence is particularly relevant as PI-PLC?1 is known to localize in the same domains. Many of the identified proteins are involved in cell cycle, proliferation and transcriptional control. In particular, many of the proteins are components of the spliceosome multi-complex, strengthening the idea that PI-PLC?1b is involved in mRNA processing and maturation. Future work will aim to better characterize the regulatory role of PI-PLC?1b in mRNA splicing.