| Popis: |
Phospholipase D2 (PLD2) is a cell membrane lipase that catalyzes the break down of phosphatidyl choline (PC) to phosphatidic acid (PA) and choline. PA is one of the major lipid second messengers that modulate a wide variety of signaling pathways, which are crucial for various cellular functions. In addition to this, PLD2 via binding to a large number of proteins and thereby regulates them independent of its lipase activity. Whereas PLD2 is involved in plethora of cellular functions, high levels of protein and/or activity of PLD2 are reported in variety of disease conditions, such as cancer metastasis and chronic inflammation making it a potential health relevant candidate to be targeted using chemical inhibitors. Data presented in this dissertation uncovers the unique ability of PLD2 to activate the small GTPases, Rac2 and Ras, by acting as a gunanine nucleotide exchange factor (GEF). This newly discovered GEF activity of PLD2 is independent of its lipase activity. Domain architecture of PLD2 reveals that, unlike other conventional Rho GEFs, PLD2 does not have Dbl homology (DH) domain. Data presented in this thesis shows that N-terminal PX and PH domains of PLD2 are key for PLD2-GEF activity. Further mutational analysis based on the multiple sequence alignments with known GEFs identified 5 amino acids, F107, F129, L166, R172 and L173 in the PX domain that are crucial for PLD2-GEF activity. In addition, GEF assays and binding experiments showed that the PH domain has to be intact to hold the substrate GTPase in proximity to the PX domain in order for PLD2-GEF to be active. This study also focused on the intermolecular and phosphorylation-dependent regulation of PLD2. Rac2-GTP, one of the PLD2-GEF products, has a dual effect on PLD2 lipase activity. Data presented here demonstrated the dual effect of lipase activity product, PA on PLD2-GEF activity unveiling the temporal intermolecular regulation of PLD2. Furthermore the data presented in this dissertation identified a potent regulatory kinase, JAK3 as a differential regulator of dual (lipase and GEF) activities of PLD2. JAK3 has a negative effect on PLD2-GEF, however it positively regulates PLD2-lipase. Coordinated control of lipase and GEF activities occur in a temporal fashion, involving both intermolecular as well as upstream regulators such as, JAK3 and switches between the two activities. Altogether these studies have contributed to the discovery of a novel lipase-GEF, PLD2 and the differential regulation of these dual activities. This provides insight into how PLD2 functions as a multifaceted signaling protein and has opened a new area of research in phospholipid biology. |
