Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction

Abstract

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X n ‫؍‬ 3-7 )-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase II␣. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003376, 1-15, 2011. Phosphoinositides (PIs)1 are lipid second messengers unique among phospholipids: Their inositol head group is rapidly phosphorylated by specific lipid kinases yielding seven distinct biologically relevant phosphatidylinositol derivatives. The coordinated activities of the PI-specific kinases and phosphatases generate PI profiles, which contribute to downstream signaling events regulating a variety of cellular processes such as proliferation, cell survival, migration, and vesicular trafficking (1-4). Impairment of PI metabolism is associated with cancer as well as neurological and immunological disorders (5-7). PIs are not only substrates for the generation of second messengers but are also second messengers themselves. They have emerged as sensors for specific PI-binding domains present in a diverse array of proteins: PH (pleckstrin homology), epsin N-terminal homology, FYVE (Fab1, YOTB, Vac1, EEA1), Phox homology, PHD (plant homeodomain), PDZ domains as well as unstructured lysine/ arginine-rich patches. These domains display a range of heterogeneity in terms of their specificity for the different PIs (8 -10) and recruit target, domain-containing, effector proteins in a temporal and spatial manner to sites of PI synthesis at many cytoplasmic locations (11).PIs (notably phosphatidylinositol(3)phosphate (PtdIns(3)P), PtdIns(4)P, PtdIns(5)P, PtdIns(4,5)P 2 , PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 ) have also been iden...

Functional Proteomics: Mapping Lipid-Protein Interactomes

“…These interactions were all validated by lipid pull down and Western immunoblotting. PIs are also found in the nucleus (Irvine 2003;Hammond et al 2004;Ye & Ahn 2008;Keune et al 2011) and we have established a quantitative and proteomic method to identify PtdIns(4,5)P 2 interacting proteins to gain insight into the PI-mediated nuclear functions (Lewis et al 2011). The workflow of the method is schematised in Figure 3.…”

“…After the isolation of www.intechopen.com . Quantitative characterisation of nuclear PI interactome by combining isotopic labelling of cells, affinity capture of proteins using PI matrices and mass spectrometry (Lewis et al 2011): C 13 K/R-labelled and C 12 K/R-labelled nuclei were incubated with 5 mM neomycin. Displaced proteins were pulled down at equal concentration with control beads or PtdIns(4,5)P 2 (PIP2)-conjugated beads.…”

“…Although extensive datasets are now available from PI interactomes in yeast and mammalian cell lines, the overlap between the PI interactomes determined in these different species is unknown. We have therefore attempted to compare the dataset obtained from PtdIns(4,5)P 2 nuclear interactome studies (Lewis et al 2011) to the datasets obtained in S. cerevisiae. Using InParanoid 7 (Ostlund et al 2010), 18 yeast orthologues were recovered from the 34 murine proteins reported in the PtdIns(4,5)P 2 nuclear interactome dataset.…”

“…In contrast, none of the 18 orthologues were found to be common to the dataset obtained from the lipid array screen by Gavin and colleagues (Gallego et al 2010) and this may be explained by the following points. Firstly, the majority of proteins chosen in the lipid array screen were known to harbour at least one lipid binding domain (LBD) as defined by the online tools for protein domain assignment SMART, Pfam or SuperFamily, whereas none of the identified nuclear proteins harboured such domains but rather simple basic amino acid rich patches (Lewis et al 2011). Secondly, the proportion of proteins annotated to the nucleus compartment analysed in the lipid array screen is not known.…”

“…List of genes found in common between the datasets obtained from the mammalian PtdIns(4,5)P 2 nuclear interactome (Lewis et al 2011) and the yeast PI interactome (Zhu et al 2001). Datasets that were compared include all yeast proteins found to bind PIs in the study by Zhu et al and 18 yeast orthologues retrieved from the 34 murine proteins identified in the PtdIns(4,5)P 2 nuclear interactome.…”