Nuclear Phosphatidylinositol 3,4,5-Trisphosphate Interactome Uncovers an Enrichment in Nucleolar Proteins

Abstract

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.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer

“…Instead, the nuclear PtdIns(3,4,5)P 3 interactome exhibited enrichment for polybasic K/R- or S/R-rich domains. Our data are consistent with a recent study that utilized the electrostatic interaction between neomycin and the phosphate group of PtdIns(3,4,5)P 3 , coupled with MS, to characterize PtdIns(3,4,5)P 3 -binding proteins for nuclear extracts from HELA cells, which similarly found an enrichment for polybasic motifs ( 63 ). One potential limitation of our PtdIns(3,4,5)P 3 interactome study is nonspecific capture of proteins by Affi-Gel-10 beads, although we performed preincubation with blank beads to remove nonspecific binders with minimal residual protein detected in respective controls.…”

Viruses and phospholipids: Friends and foes during infection

“…12,13,20,78 Nuclear PI (4,5)P 2 regulates the enzymatic activity of a number of nuclear proteins and conversely, a plethora of nuclear proteins bind PI(4,5)P 2 . [14][15][16][17][18]87 Other nuclear PIPs found in the nucleoplasm or in nuclear speckles include PI(3,4)P 2 , 20 PI(3,4,5)P 3 found in the nucleolus, 88 or PI(4)P found in the nuclear periphery, nucleoplasm and nuclear speckles. 19,20 The identification of nuclear proteins containing phosphoinositide interaction domains strongly suggests that they are important regulators of DNA topology, chromatin conformation, and RNA maturation and export.…”

Cell-Permeable Fluorescent Sensors Enable Rapid Live Cell Visualization of Plasma Membrane and Nuclear PIP3 Pools

“…25 Importantly, the organization of PIP3 in nonmembranous environments within the nucleus, especially the nucleolus, is unclear. 27,31 It is hypothesized that the acyl chains might be sheltered from the aqueous environment, but this has not been demonstrated thus far. 27,31 In this backdrop, cell-permeable, fluorescent chemical sensors for imaging and tracking PIP3 can provide key information on the fundamental basis of PIP3-mediated cell-signaling pathways and importantly provide the yet unavailable handle for studying the nuclear pool.…”

“…4 This poses a challenge for imaging the nuclear PIP3 pool since PI (3,4)P2 is also present within the nucleus. 27 In this study, we have developed two short-peptide-based, environment-sensitive, ratiometric fluorescent sensors, MFR-K17H and DAN-NG-H12G, that rapidly enter the cell cytoplasm and orthogonally target and image the plasma membrane and nuclear PIP3 pools, respectively. MFR-K17H exhibited up to 45 times increase in visible fluorescence emission in the presence of 6.5 μM PIP3 with a limit of detection of 193 nM.…”

“…While the roles of PIP3 in the cell membrane are reasonably well-established and putative concentrations estimated, the nuclear PIP3 pool has remained elusive. ,, Anti-PIP3 antibody staining studies have provided definite evidence for the presence of PIP3 within nucleoli . Recent interactomics studies showed that nuclear PIP3 interacted with proteins enriched in RNA and DNA binding domains . These proteins spanned functions including RNA processing, cytokinesis, protein folding, and DNA repair. , Proteins involved in cell survival and proliferation, like PI3Ks, were also identified .…”