A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha.
Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-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 structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-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 Ca(2+) 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 DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), 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 Ca(2+)-induced DGK alpha activation.
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Y
Y
Jiang
W
W
Qian
J W
JW
Hawes
J P
JP
Walsh
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D019852:Diacylglycerol Kinase; D002118:Calcium