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...

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We reveal the identification of a polybasic motif necessary for polyphosphoinositide interaction and nucleolar targeting of ErbB3 binding protein 1 (EBP1). EBP1 interacts directly with phosphatidylinositol(3,4,5)-triphosphate and their association is detected in the nucleolus, implying regulatory roles of nucleolar processes.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p < 0.001) and metastatic lesion (p = 0.010). Our results support the notion that the PI3K signaling pathway might be activated, both dependent- and independently of PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.

The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.