The Pleckstrin Homology Domain of Diacylglycerol Kinase η Strongly and Selectively Binds to Phosphatidylinositol 4,5-Bisphosphate

Abstract

Type II diacylglycerol kinase (DGK) isozymes (␦, , and ) 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 DGK showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P 2 ) 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)P 2 binding activity of the DGK-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) ␦1, which is generally utilized as a cellular PI(4,5)P 2 -probe, the DGK-PH is equal to or superior than the PLC␦1-PH in terms of affinity and selectivity for PI(4,5)P 2 . Furthermore, in COS-7 cells, GFP-fused wild-type DGK1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLC␦1-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 DGK-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P 2 , were indeed unable to bind to PI(4,5)P 2 and co-localize with the PLC␦1-PH even in osmotically shocked cells. Overexpression of wild-type DGK1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGK-PH preferentially interacts with PI(4,5)P 2 and has crucial roles in regulating the subcellular localization and physiological function of DGK. Moreover, the DGK-PH could serve as an excellent cellular sensor for PI(4,5)P 2 . Diacylglycerol kinase (DGK)2 phosphorylates diacylglycerol to produce phosphatidic acid (1-5). Diacylglycerol (6 -8) and phosphatidic acid (PA) (9, 10) are well recognized as lipid second messengers. DGK appears to participate in various physiological events by modulating the balance between the two bioactive lipids diacylglycerol and phosphatidic acid (1-5). Ten mammalian DGK isozymes (␣, ␤, ␥, ␦, ⑀, , , , , and ) containing two or three characteristic zinc finger-like C1 domains and a common catalytic region are divided into five groups (types I-V) according to their structural features (1-5).Type II DGKs (11) consist of DGK␦ (12), - (13), and -(14). Moreover, alternative splicing products of DGK␦ (␦1 and ␦2) (15) and -(1 and 2) (16) have been identified. Type II DGKs have a pleckstrin homology domain (PH) in common at their N termini and a catalytic domain that is divided into two subdomains (catalytic subdomains a and b). DGKs ␦1, ␦2, and 2, but not DGKs 1 and , contain a sterile ␣-motif domain at their C termini. It has been demonstrated that DGKs ␦1, ␦2, and 2 form oligomers through interactions between their sterile ␣-motif domains and that this oligomer formation regulates the activities and subcellular localization of these DGK isoforms (15)(16)(1...

Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4

“…Thus, we tested whether DGKη3 and η4 respond to osmotic stress stimulation. DGKη1 and η2 were translocated to punctate vesicles in the cytoplasm in response to osmotic stress, as previously reported [ 12 , 13 , 26 , 29 ]. In NEC8 cells DGKη1 or η4 were also osmotic stress-dependently translocated to punctate vesicles in the cytoplasm ( Fig 5 ).…”

“…All of the type II DGK isoforms possess a pleckstrin homology domain at their N termini and a separated catalytic domain, and DGKs δ1, δ2 and η2 but not DGKs η1 or κ contain a sterile α-motif (SAM) domain at their C termini. The pleckstrin homology domain of DGKη was found to preferentially interact with phosphatidylinositol-4,5-bisphosphate [ 13 ]. Moreover, it has been reported that DGKs δ1, δ2 and η2 formed oligomers through interactions among their SAM domains and that this oligomer formation regulates the subcellular localizations of these DGK isoforms [ 11 , 12 , 14 – 16 ].…”

Deficiency of diacylglycerol kinase η induces lithium‐sensitive mania‐like behavior

“…Moreover, we recently found that the pleckstrin domain of DGKη is strongly bound to phosphatidylinositol 4,5‐bisphosphate, a product of the phosphatidylinositol turnover (Kume et al . ). We also revealed that DGKη is a unique enzyme with high affinity for DG (Komenoi et al .…”

Characterization of α‐synuclein N‐terminal domain as a novel cellular phosphatidic acid sensor

“…pEGFP‐DGKβ‐WT, pEGFP‐DGKβ‐KD (G495D), pEGFP‐DGKγ‐WT, pEGFP‐DGKγ‐KD (G494D), and pDsRed‐monomer‐PLCδ1‐PHD were previously generated . pAcGFP‐α‐Syn‐N and pDsRed‐monomer‐α‐Syn‐N (Met1–Lys60) were constructed by inserting PCR fragments encoding Met1‐Lys60 from the pET‐14b‐α‐Syn into the EcoRI/SalI sites of pAcGFP1‐C1 or the pDsRed‐monomer‐C1 vector (Takara‐Clontech, Kusatsu, Japan).…”