Systems biology studies for understanding the roles of phospholipase D and phosphatidic acid

Abstract

Phospholipase D (PLD) hydrolyzes phosphatidylcholine (PC) to generate choline and phosphatidic acid (PA), which acts as a second messenger. PLD activity can be regulated by dynamic interactions with multiple binding proteins and phospholipids. Furthermore, PA is known to bind a variety of proteins to mediate cellular functions such as cell growth, proliferation, differentiation, migration, and cytoskeletal reorganization. Although many binders for PLD and PA have been identified and characterized individually and the multifunctional roles of PLD/PA have been reported, any systemic analysis at the network level to explain common mode and mechanism of PLD signaling has not been studied yet. To start the system biology analysis for PLD/PA signaling systems, I first collected and integrated the information about PLD and its binding partners from several biological database. Next, I reconstructed an interaction network for PLD and PA in which they serve as signaling hubs. Although PLDs are highly expressed in several type of cancers, the functional roles and mechanisms of PLD/PA signaling for the cancer progression are poorly understood. To provide the systemic basis for the understanding of PLD/PA signaling in cancer, I analyzed mRNA expression level of PLD and its binding partners in ten cancer types. PLD1 showed higher cancer-associated deregulation than PLD2, and PLD/PA-interacting molecules also are notably deregulated in the cancers. Analysis of PLD signaling network suggests that PLD and PA may act as signaling coordinators for the proliferation in cancer. PLD/PA interaction networks can be decomposed into smaller units known as ‘network motifs’, which is a small recurring regulation patterns of interaction that can carry out specific information-processing function. I identified several network motifs including 24 feedback and 129 feed-forward loops in the PLD/PA interaction network. Furthermore, I found that PLD and PA form feed-forward loops involving GTPases (such as Rheb, Arf, and RhoA) and kinases (including mTORC1, PI5K, and PKC), and defined ‘GPK signaling modules’. By using a systems approach, I suggest that PLD could achieve functionality the filtering out of noise-like transient signals via the GPK signaling modules. However, the verification of GPK models in a cell system remains a substantial challenge. Therefore, to suggest the GPK modules as a functional signaling systems in cells, I experimentally validated the models in the mTORC1 signaling. In this study, I found the coordination mechanism that PA regulate mTORC1 signaling through the control of the Rheb-binding to mTOR kinase. This finding provide answers to the long-standing question of how Rheb and PA activate mTORC1 signaling. Furthermore, I also found that PA is key factor that regulate the delay time and amplitude of mTORC1 activation. These results suggest that mTORC1 signaling module is a functional GPK module that can filter out noise signals. In conclusion, this study is a first approach to investigate the roles of PLD and PA at the network level. Here, I suggests that PLD and PA may plays critical roles as a dynamic signaling coordinator in complex biological system via systems approaches such as network analysis. I believe that this study offers us opportunities for gaining the novel insights of regulation and functions of PLD, and collected information in this study will be useful for further study in PLD research filed. Last, this study show that systems biology can be used to understand the fundamental principles of cell signaling.