title: EF-hand motifs of diacylglycerol kinase α interact intra-molecularly with its C1 domains.
abstract: Diacylglycerol kinase (DGK) α, which is activated by Ca(2+), contains a recoverin homology (RVH) domain, tandem repeats of two Ca(2+)-binding EF-hand motifs, two cysteine-rich C1 domains and the catalytic domain. We previously found that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active and that the N-terminal region of DGKα, consisting of the RVH domain and EF-hand motifs, interacted intra-molecularly with the C-terminal region containing the C1 and catalytic domains. In this study, we narrowed down the interaction regions of DGKα. At the C-terminal region, the C1 domains are responsible for the intra-molecular interaction. At the N-terminal region, the EF-hand motifs mainly contribute to the interaction. Moreover, using highly purified EF-hand motifs and C1 domains, we demonstrate that they directly bind to each other. The co-precipitation of these two domains was clearly attenuated by the addition of Ca(2+). These results indicate that the Ca(2+)-induced dissociation of the intra-molecular interaction between the EF-hand motifs and the C1 domains of DGKα is the key event that regulates the activity of the enzyme.
authors.affiliation: Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Sakai
authors.affiliation: Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Sakane
keywords: C1 domain; C1Ds, C1 domains; CR, catalytic region; Calcium; DG, diacylglycerol; DGK, diacylglycerol kinase; Diacylglycerol kinase; EF-hand; EFHs, EF-hand motifs; EGFP, enhanced green fluorescence protein; EGTA, ethylene glycol tetraacetic acid; GST, glutathione S-transferase; Intra-molecular interaction; RVH, recoverin homology; RVHD, RVH domain; TF, trigger factor