title: Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1.
abstract: Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.
authors.affiliation: Department of Pathology, Northwestern University, Chicago, Illinois 60611, United States. j-lomasney@northwestern.edu
authors.forename: Jon W
authors.initials: JW
authors.lastname: Lomasney
authors.forename: Hwei-Fang
authors.initials: HF
authors.lastname: Cheng
authors.forename: Minae
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Klim
authors.initials: K
authors.lastname: King
mesh_terms: D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism*; D054803:Phospholipase C delta / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008024:Ligands; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D001224:Aspartic Acid; D054803:Phospholipase C delta; D002118:Calcium