The Ser313 of AP180 regulates phospholipase D1 (PLD1) activity through binding to second phosphodiesterase domain of PLD1

Abstract

Phospholipase D (PLD) hydrolyses phosphatidylcholine to phosphatidic acid (PA) and choline. PLD acts as a survival factor against death signal in several cancer cells. This study tried to elucidate how inhibitory mechanisms of PLD activity occurred. According to previous studies, clathrin assembly proteins such as amphiphysins, synaptojanin, and AP180 can inhibit PLD activity. To begin the investigation where is a critical motif in AP180 for binding with PLD and inhibition of PLD activity. Human AP180 gene was cloned from human cDNA library, and inhibition of phobol-12-myristate 13-acetate (PMA) induced PLD activity by hAP180 transfection was detected. By performing hAP180 deletion series study, the 11ea amino acids from Pro-310 to Lys-320 were important residues for binding with PLD1. Among these amino acids, five peptides (The310 ~ Pro314) in C-terminal part were shown as a critical region for the inhibition of PLD activity when treated synthetic penta or hepta peptide into both of KATOIII and SiHa cells. Next we constructed point mutated mutants of every five peptide from The310 to Pro314 into Ala. Thr312, Ser313, and Pro314 of hAP180 were critical amino acids for the inhibition of PLD activity. Among these three amino acids, Ser313 was found to be the most important amino acid for the inhibition of PLD activity. This result is further supported by PLD assay and immunoprecipitation study. Furthermore we also found that second phosphodiesterase domain in PLD1 is important to binding with AP180. These results indicated that Thr312 ~ Pro314 (especially Ser313) of AP180 can regulate PLD1 activity through binding with second phosphodiesterase domain of PLD1. Taken together, these results promise for the possibility of developing peptido-mimetic drug which can inhibit PLD activity. If these amino acids sequences will be applied to synthesis of a therapeutic peptido-mimetic drug, PLD-related survival signal in cancer will be under control.