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.
We previously reported that diacylglycerol kinase β (DGKβ) induces neurites and branches, contributing to higher brain function including emotion and memories. However, the detailed molecular mechanism of DGKβ function remains unknown. Therefore, we constructed various mutants of DGKβ and compared their enzyme activity, intracellular localization, and ability to induce neurites and branching in SH-SY5Y cells. Even when RVH-domain and EF-hand motif were deleted, the mutant showed similar plasma membrane localization and neurite induction compared to wild type (WT), although the kinase activity of the mutant was three times higher than that of WT. In contrast, further deletion of C1 domain reduced the activity to 50% and abolished plasma membrane localization and neurite induction ability. When 34 amino acids were deleted from C-terminus, the mutants completely lost enzyme activity, plasma membrane localization, and the ability to induce neurites. A kinase-negative mutant of DGKβ retained plasma membrane localization and induced significant neurites and branches; however, the rate of induction was weaker than that of WT. Furthermore, C1A and C1B mutants, which have a mutation in a cysteine residue in the C1A or C1B domain, and the RK/E mutant, which has substitutions of arginine and lysine to glutamic acid in a cluster of basic amino acids at the C-terminus, lost their plasma membrane localization and neurite induction ability. These results indicate that in addition to kinase activity, plasma membrane localization via the C1 domain and basic amino acids at the C-terminus were indispensable for neurite induction by DGKβ.
Laboratory of Chemistry and Utilization of Animal Production Resources, Department of Applied Chemistry in Bioscience, Graduate School of Agricultural Science, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan.
Takuya
T
Kano
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Takeshi
T
Kouzuki
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Satoru
S
Mizuno
Laboratory of Chemistry and Utilization of Animal Production Resources, Department of Applied Chemistry in Bioscience, Graduate School of Agricultural Science, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan.
Shuji
S
Ueda
Laboratory of Chemistry and Utilization of Animal Production Resources, Department of Applied Chemistry in Bioscience, Graduate School of Agricultural Science, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan.
Minoru
M
Yamanoue
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Fumio
F
Sakane
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Naoaki
N
Saito
Laboratory of Chemistry and Utilization of Animal Production Resources, Department of Applied Chemistry in Bioscience, Graduate School of Agricultural Science, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan. Electronic address: shirai@kobe-u.ac.jp.
Yasuhito
Y
Shirai
D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats
D024361:Amino Acids, Basic; D019852:Diacylglycerol Kinase
Branching; Diacylglycerol kinase; Neurite outgrowth