The beta-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGK beta remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGK beta in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGK beta mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGK beta. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGK beta selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, beta 2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with alpha 2-chimaerin, but not with alpha 1- or beta 1-chimaerin. Moreover, DGK beta enhanced the plasma membrane localization of beta 2-chimaerin. These results suggest that DGK beta plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation. (C) 2018 Elsevier Inc. All rights reserved.