Calcium negatively regulates an intramolecular interaction between the N-terminal recoverin homology and EF-hand motif domains and the C-terminal C1 and catalytic domains of diacylglycerol kinase α.
The type I diacylglycerol kinase (DGK) isozymes (α, β and γ) 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 DGKα mutant lacking the RVH domain and EF-hands was constitutively active, implying that the N-terminal region (NTR) of DGKα, 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 DGKα-NTR construct physically binds to a green fluorescent protein (GFP)-fused DGKα-CTR construct. Moreover, co-precipitation of GFP-DGKα-CTR with GST-DGKα-NTR was clearly attenuated by the addition of 1 μM Ca2+. This result indicates that Ca2+ induces dissociation of the physical interaction between DGKα-NTR and DGKα-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 α-helical content of DGKα-NTR. These results suggest that Ca2+-induced conformational changes in the NTR release the intramolecular association between the NTR and the CTR of DGKα.
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Masato
M
Takahashi
Tatsuya
T
Yamamoto
Hiromichi
H
Sakai
Fumio
F
Sakane
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D013552:Swine
D019852:Diacylglycerol Kinase; D002118:Calcium