The Pleckstrin Homology Domain of Diacylglycerol Kinase η Strongly and Selectively Binds to Phosphatidylinositol 4,5-Bisphosphate.
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.
['Kume|Aiko|A|', 'Kawase|Koki|K|', 'Komenoi|Suguru|S|', 'Usuki|Takako|T|', 'Takeshita|Ena|E|', 'Sakai|Hiromichi|H|', 'Sakane|Fumio|F|']
D000595:Amino Acid Sequence D000818:Animals D019556:COS Cells D002522:Chlorocebus aethiops D019852:Diacylglycerol Kinase D006801:Humans D020935:MAP Kinase Signaling System D008969:Molecular Sequence Data D009997:Osmotic Pressure D019269:Phosphatidylinositol 4,5-Diphosphate D011485:Protein Binding D017434:Protein Structure, Tertiary D016415:Sequence Alignment
pleckstrin homology domain diacylglycerol diacylglycerol kinase phosphatidylinositol 4,5-bisphosphate cancer extracellular signal-regulated kinase (ERK) inositol phospholipid lipid-binding protein phosphatidylinositol signaling