| dc.description.abstract |
Phospholipase D2 (PLD2) is a lipase that catalyzes the breakdown of phopsphatidyl choline to phosphatidic acid (PA) which in turn is involved in variety of cell signaling pathways. PLD2 also interacts with many proteins like Grb2, Rac2 and actin etc. through which it regulates actin cytoskeleton. Therefore it is one of the key players in both physiological and pathological scenarios by mediating cell migration and cell invasion respectively. In addition to its lipase activity and its ability to interact with other proteins, surprisingly our lab for the first time discovered that it is a guanine nucleotide exchange factor (GEF). Since it is a newly identified GEF very little is known about its GEF activity. The objective of the present study is to identify the key aminoacids involved in the GEF activity of PLD2 and also the upstream regulators. With multiple sequence alignment of PLD2 and known GEFs, Dbs and Tiam1, conserved regions amongst the three proteins were manually derived. Candidate aminoacids in the conserved regions were selected to perform further mutational analysis to identify the aminoacids involved in GEF activity. In addition, chemotaxis and phagocytosis assays were performed with macrophage cell lines to confirm the functional relevance of the mutants. We showed that the PX and PH domains are the GEF activity containing domains, PX being the important. Within the PX domain, we propose that the α-helix-3 has putative GEF activity site. The mutants that are deficient in GEF activity are also found to be defective in performing chemotaxis and phagocytosis. With respect to the regulation of GEF activity our preliminary data suggests that JAK3, a tyrosine kinase that is known to phosphorylate at tyrosine 415 of PLD2, inhibits GEF activity. In future we would like to investigate other upstream regulators and how lipase and GEF activities are differentially regulated. |
|
