DELETION AND SITE-DIRECTED MUTAGENESIS OF EF-HAND DOMAIN OF PHOSPHOLIPASE C-DELTA-1 - EFFECTS ON ITS ACTIVITY

In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-FI), deletion and point mutation of the e nzyme were performed and the mutated cDNAs were expressed in CHO cells and E. coli AD202 strain. Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity. However, the decreas ed PLC activity to 15-20% by deletion of the EF-hand motif (144-172) w as still Ca2+-dependent. Furthermore, mutants, in which conserved Asp( 153), Asp(157), Glu(164) or all these acidic amino acids in the EF-han d motif were replaced with alanine residues, showed nearly the same PL C activity and Ca2+-dependency as those of wild-type. These results su ggest that the region containing the EF-hand motif may not play a role in regulation of Ca2+-sensitivity of PLC-delta 1, but is important fo r its activity. (C) 1995 Academic Press, Inc.