A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase α

Ying Jiang, Weijun Qian, John W. Hawes, James P. Walsh

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase α is an 82-kDa DGK isoform that is activated in vitro by Ca2+. The DGKα regulatory region includes tandem C1 protein kinase C homology domains and Ca2+-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the strucrural basis of Ca2+ regulation, we expressed a series of DGKα deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca2+-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca2+ to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGKα fragment that included both the RVH domain and EF hands. These observations suggested that Ca2+ elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGKα. As with Ca2+, this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca2+-induced DGKα activation.

Original languageEnglish (US)
Pages (from-to)34092-34099
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number44
DOIs
StatePublished - Nov 3 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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