Polyphosphoinositides (PPIns) play essential roles as lipid signaling molecules, and many of their functions have been elucidated in the cytoplasm. However, PPIns are also intranuclear where they contribute to chromatin remodeling, transcription, and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5) P 3 ), has been mapped to the nucleus and nucleoli, but its role remains unclear in this subcellular compartment. To gain further insights into the nuclear functions of PtdIns(3,4,5) P 3 , we applied a previously developed quantitative MS-based approach to identify the targets of PtdIns(3,4,5) P 3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5) P 3 -interacting partners, and gene ontology analysis for the biological functions of this dataset revealed an enrichment in RNA processing/splicing, cytokinesis, protein folding, and DNA repair. Interestingly, about half of these interactors were common to nucleolar protein datasets, some of which had dual functions in rRNA processes and DNA repair, including poly(ADP-ribose) polymerase 1 (PARP1, now referred as ADP-ribosyltransferase 1). PARP1 was found to interact directly with PPIn via three polybasic regions in the DNA-binding domain and the linker located N-terminal of the catalytic region. PARP1 was shown to bind to PtdIns(3,4,5) P 3 as well as phosphatidylinositol 3,4-bisphosphate in vitro and to colocalize with PtdIns(3,4,5) P 3 in the nucleolus and with phosphatidylinositol 3,4-bisphosphate in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5) P 3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5) P 3 -mediated interactions in the nucleus and nucleolus.

The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared to PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are however unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA levels in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear/cytoplasmic staining ratio were detected in high grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) were measured in the nucleus of EC cells and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC.

Polyphosphoinositides (PPIn) play essential functions as lipid signalling molecules and many of their functions have been elucidated in the cytoplasm. However, PPIn are also intranuclear where they contribute to chromatin remodelling, transcription and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has been mapped to the nucleus and nucleoli but its role remains unclear in this subcellular compartment. To gain further insights into the nuclear functions of PtdIns(3,4,5)P3, we applied a previously developed quantitative mass spectrometry-based approach to identify the targets of PtdIns(3,4,5)P3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5)P3-interacting proteins and gene ontology analysis for the biological functions of this dataset revealed an enrichment in RNA processing/splicing, cytokinesis, protein folding and DNA repair. Interestingly, about half of these interactors were common to nucleolar protein datasets, some of which had dual functions in rRNA transcription and DNA repair, including Poly(ADP-Ribose) Polymerase 1 (PARP1/ARTD1). PARP1 was found to interact directly with PtdIns(3,4,5)P3 as well as PtdIns(3,4)P2 and to co-localise with PtdIns(3,4,5)P3 in the nucleolus and with PtdIns(3,4)P2 in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5)P3 interactome reported here identified several nucleolar proteins and further pointed to roles for this lipid in these processes.

No abstract

Genes encoding for components of the phosphoinositide 3-kinase (PI3K) pathway are frequently mutated in cancer, including inactivating mutations of PTEN and activating mutations of PIK3CA, encoding the PI3K catalytic subunit p110α. PIK3CB, encoding p110β, is rarely mutated, but can contribute to tumourigenesis in some PTEN-deficient tumours. The underlying molecular mechanisms are however poorly understood. By analysing cell lines and annotated clinical samples, we have previously found that p110β is highly expressed in endometrial cancer (EC) cell lines and that PIK3CB mRNA levels increase early in primary tumours correlating with lower survival. Selective inhibition of p110α and p110β led to different effects on cell signalling and cell function, p110α activity being correlated to cell survival in PIK3CA mutant cells and p110β with cell proliferation in PTEN-deficient cells. To understand the mechanisms governing the differential roles of these isoforms, we assessed their sub-cellular localisation. p110α was cytoplasmic whereas p110β was both cytoplasmic and nuclear with increased levels in both compartments in cancer cells. Immunohistochemistry of p110β in clinically annotated patient tumour sections revealed high nuclear/cytoplasmic staining ratio, which correlated significantly with higher grades. Consistently, the presence of high levels of p110β in the nuclei of EC cells, correlated with high levels of its product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in the nucleus. Using immunofluorescence labelling, we observed both p110β and PtdIns(3,4,5)P3 in the nucleoli of EC cell lines. The production of nucleolar PtdIns(3,4,5)P3 was dependent upon p110β activity.EC cells with high levels of nuclear PtdIns(3,4,5)P3 and p110β showed elevated nucleolar activity as assessed by the increase in 47S pre-rRNA transcriptional levels in a p110βdependent manner. Altogether, these results present a nucleolar role for the PI3K pathway that may contribute to tumour progression in endometrial cancer.