Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain.
Cytosolic phospholipase A2 (cPLA2) associates with natural membranes in response to physiological increases in Ca2+, resulting in the selective hydrolysis of arachidonyl phospholipids. The isolation and sequence analysis of cPLA2 cDNA clones from four different species revealed several highly conserved regions. The NH2-terminal conserved region is homologous to several other Ca(2+)-dependent lipid-binding proteins. Here we report that the first 178 residues of cPLA2, containing the homologous Ca(2+)-dependent lipid-binding (CaLB) motif, and another recombinant protein containing the cPLA2(1-178) fragment placed at the COOH terminus of the maltose-binding protein (MBP-CaLB) associate with membranes in a Ca(2+)-dependent manner. cPLA2 and MBP-CaLB also bind to synthetic liposomes at physiological Ca2+ concentrations, demonstrating that accessory proteins are not required. In contrast, delta C2, a truncated cPLA2 lacking the CaLB domain, fails to associate with membranes and fails to hydrolyze liposomal substrates. However, both delta C2 and cPLA2 hydrolyze monomeric 1-palmitoyl-2-lysophosphatidylcholine at identical rates in a Ca(2+)-independent fashion. These results delineate two functionally distinct domains of cPLA2, the Ca(2+)-independent catalytic domain, and the regulatory CaLB domain that presents the catalytic domain to the membrane in response to elevated Ca2+.
Genetics Institute, Small Molecule Drug Discovery Group, Cambridge, Massachusetts 01240.
E A
EA
Nalefski
L A
LA
Sultzman
D M
DM
Martin
R W
RW
Kriz
P S
PS
Towler
J L
JL
Knopf
J D
JD
Clark
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D005399:Fishes; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium