The molecular mechanisms of hAP180 for inhibition of PLD activity

초록

According to recent studies, phospholipase D (PLD) is a potent candidate for the development of new class of anticancer drug which can enhance anticancer drug efficacy or cancer cell sensitivity, since PLD acts as a survival factor during anticancer drug induced cell death. We also reported that PLD suppresses taxotere induced apoptosis in stomach cancer cells. PLD metabolism is implicated in various physiological processes including membrane trafficking, cytoskeletal reorganization, cell proliferation, differentiation, survival, and apoptosis. PLD is an ubiquitous enzyme which catalyzes the hydrolysis of phosphatidyl choline (PC) to phosphatidic acid (PA) and choline. PLD has two isoforms, PLD1 and PLD2, and they differ in the mechanism of activation and subcellular localization. Three molecules that can inhibit PLD activity were reported previously. Fist, synaptojanin inhibits PLD activity by hydrolysis of PI(4,5)P2. ohters, amphiphysin and assembly protein 180 (AP180, also known as NP185, pp155, F1-20, and AP3), block PLD activity through binding to PLD. Basically, AP180 is supposed to play an important role in the first step of clathrin-coated pit initiation in that it tethers clathrin to the plasma membrane. Based on these backgrounds, hAP180 was cloned from human cDNA library, since this study focused on human cancers, specifically including human stomach cancer cell (KATOIII) and human cervical cancer cells (SiHa). In this study, it was hypothesized that hAP180 regulates or inhitbits PLD activity, thus the molecular mechanisms and structural specific motif in hAP180 on inhibition of PLD activity were investigated through PLD assay, immunoprecipitation and site directed point mutagenesis. The results from this study suggest that hAP180 can control and inhibit PLD activity which is the same as rat AP180 (rAP180) possibly through inhitition of Bcl-2 expression resulting in increasing sensitivity of cancer cell against anticancer agents. It is the most remarkerble result that amino acids from Thr to Pro are responsible not only for binding to PLD but also for inhibition of PLD activity. On top of these results, the three amino acids -- Thr, Ser, and Pro are important for the inhibition of PLD activity. Moreover, among these amino acids, Ser is a critical amino acid for binding to PLD1.