We previously reported that diacylglycerol kinase 13 (DGK beta) induces neurites and branches, contributing to higher brain function including emotion and memories. However, the detailed molecular mechanism of DGK beta function remains unknown. Therefore, we constructed various mutants of DGK beta 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 Cl 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 DGK beta retained plasma membrane localization and induced significant neurites and branches; however, the rate of induction was weaker than that of WT. Furthermore, CIA and C1B mutants, which have a mutation in a cysteine residue in the CIA 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 Cl domain and basic amino acids at the C-terminus were indispensable for neurite induction by DGK beta. (C) 2014 Elsevier Inc. All rights reserved.