Both the C1 domain and a basic amino acid cluster at the C-terminus are important for the neurite and branch induction ability of DGKβ
Introduction
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DG is an important lipid messenger that regulates several proteins, including protein kinase C (PKC), chimerins and Unc-13. PA is also an important signaling molecule that activates several enzymes, including the mammalian target of rapamycin (mTOR) and a typical PKC. Therefore, DGK is thought to be a key enzyme for numerous cellular responses [1], [2], [3], [4], [5]. Indeed, recent researches using DGK knockout (KO) mice clearly demonstrated importance of DGK in the immune system [6], [7], its pathophysiological roles in brain and heart [7], and its role in insulin resistance in diabetes [8], [9], [10].
To date, ten subtypes of DGK have been cloned and categorized into five groups based on primary structural motifs. All DGKs, except DGKθ, have two cysteine-rich regions homologous to those of PKCs (C1A and C1B domains). These regions are located in regulatory domain at the N-terminal half, and a catalytic domain is present in the C-terminal half. DGKθ has three C1 domains and a separated catalytic domain. In addition, Type I DGKs (DGKα, β and γ) have an EF-hand and a recoverin homology (RVH) domain. Type II DGKs (DGKδ, η and κ) have a pleckstrin homology (PH) domain instead of EF-hand and RVH domains at the N-terminus. Type III DGK (DGKε) contains only the C1 domains. Type IV DGKs (DGKζ and ι) have a myristoylated alanine-rich C-kinase substrate-like region at the N-terminus and four ankyrin repeats at the C-terminus. Type V DGK (DGKθ) has a proline- and glycine-rich and PH domains.
DGKβ was cloned from a rat brain cDNA library in 1993 [11] and is predominantly localized in neurons, specifically in the cerebral cortex, hippocampus, and caudate–putamen [11], [12], [13]. The expression of DGKβ rapidly increases after 14 days of age [12], when synaptic maturation progresses. In addition, Caricasole et al. reported that one of the splice variants of human DGKβ, which lacks 35 amino acids at the C-terminus but has an additional 4-amino-acid extension (DGKβ SV3; GenBank accession number AX032745), is associated with a human DGKβ EST that is annotated as differentially expressed in patients with mood disorders [14]. These results suggest that DGKβ is important for neuronal functions and is related to neuronal diseases. Indeed, we produced DGKβ KO mice and showed that DGKβ regulates neurites, branches, and spines, and its loss causes impairment of emotion and long-term memory related to cognitive functions [15], [16]. However, the precise molecular mechanisms of the DGKβ-induced morphological change remain unknown. Therefore, we constructed various mutants of DGKβ and compared their enzyme activity, intracellular localization, and ability to induce neurites and branches in SH-SY5Y cells.
Section snippets
Materials
SY-SY5Y and COS-7 cells were purchased from RIKEN CELL BANK. The plasmids encoding DGKβ was previously described [16]. The anti-GFP antibody was made in house.
Cell culture
COS-7 cells were cultured in DMEM medium (Nacali tesque). SH-SY5Y cells were cultured in DMEM/F-12 medium supplemented with 10% FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml) and 2 mM Glutamax. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
Construction of plasmids encoding GFP-DGKβ and mutants
The constructs encoding rat DGKβ fused to GFP (DGKβFL) were
Neurite induction ability of the DGKβ mutants
First, we examined the neurite induction ability of a series of domain deletion mutants of DGKβ (Fig. 1A). All mutants were the appropriate molecular weight without any significant degradation, although a small amount of degraded product was detected in the cases of CA and IncABS (Fig. 1B).
SH-SY5Y cells overexpressing GFP-fused full length DGKβ (DGKβFL) had more neurites and branches than control cells expressing GFP alone; statistical analysis showed that approximately 50% of the cells
Discussion
Similar to other type I DGKs, DGKα and γ, DGKβ consists of several domains, including the RVH domain, EF-hand domain, C1 domain, and kinase domain. The role of each domain in DGKβ has not been fully elucidated. Here, we obtained novel information regarding the roles of at least three regions of DGKβ on activity, localization, and neurite induction ability.
The N-terminus region, including the RVH and EF-hand domains, is thought to be a calcium ion sensor. Indeed, the kinase activity of DGKα is
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific Research (C) of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Research Grant of Uehara Memorial Foundation.
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