title: The Pleckstrin Homology Domain of Diacylglycerol Kinase η Strongly and Selectively Binds to Phosphatidylinositol 4,5-Bisphosphate.
abstract: Type II diacylglycerol kinase (DGK) isozymes (δ, η, and κ) have a pleckstrin homology domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGKη showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) among the various glycero- and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P2binding activity of the DGKη-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) δ1, which is generally utilized as a cellular PI(4,5)P2- probe, the DGKη-PH is equal to or superior than the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2 Furthermore, in COS-7 cells, GFP-fused wild-type DGKη1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLCδ1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGKη-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P2, were indeed unable to bind to PI(4,5)P2and co-localize with the PLCδ1-PH even in osmotically shocked cells. Overexpression of wild-type DGKη1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGKη-PH preferentially interacts with PI(4,5)P2and has crucial roles in regulating the subcellular localization and physiological function of DGKη. Moreover, the DGKη-PH could serve as an excellent cellular sensor for PI(4,5)P2.
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Kume
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Kawase
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Suguru
authors.initials: S
authors.lastname: Komenoi
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Usuki
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Ena
authors.initials: E
authors.lastname: Takeshita
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Sakai
authors.affiliation: From the Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan sakane@faculty.chiba-u.jp.
authors.forename: Fumio
authors.initials: F
authors.lastname: Sakane
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; C000610117:DGKH protein, human; D019852:Diacylglycerol Kinase
keywords: cancer; diacylglycerol; diacylglycerol kinase; extracellular signal-regulated kinase (ERK); inositol phospholipid; lipid-binding protein; phosphatidylinositol 4,5-bisphosphate; phosphatidylinositol signaling; pleckstrin homology domain