The type I diacylglycerol kinase (DGK) isozymes (alpha, beta and gamma) contain a shared recoverin homology (RVH) domain, a tandem repeat of Ca2+-binding EF-hand motifs, two cysteine-rich C1 domains, and the catalytic domain. We previously reported that a DGK alpha mutant lacking the RVH domain and EF-hands was constitutively active, implying that the N-terminal region (NTR) of DGK alpha, consisting of the RVH domain and EF-hand motifs, intramolecularly interacts with and masks the activity of the C-terminal region (CTR), containing the C1 and catalytic domains. In this study, we demonstrate that a glutathione S-transferase (GST)-fused DGK alpha-NTR construct physically binds to a green fluorescent protein (GFP)-fused DGK alpha-CTR construct. Moreover, co-precipitation of GFP-DGK alpha-CTR with GST-DGK alpha-NTR was clearly attenuated by the addition of 1 mu M Ca2+. This result indicates that Ca2+ induces dissociation of the physical interaction between DGK alpha-NTR and DGK alpha-CTR. In addition to previously reported calcium-dependent changes in the hydrophobicity and net surface charge, Ca2+ also appeared to induce a decrease in the alpha-helical content of DGK alpha-NTR. These results suggest that Ca2+-induced conformational changes in the NTR release the intramolecular association between the NTR and the CTR of DGK alpha. (C) 2012 Elsevier Inc. All rights reserved.