A GEF-to-phospholipase molecular switch caused by PA, RAC and jak tyrosine kinase, that explains leukocyte cell migration

Abstract

SummaryPhospholipase D2 (PLD2) is a cell-signaling molecule that bears two activities: a guanine-nucleotide exchange factor (GEF) and a lipase that reside in the PX/PH domains and in two HKD domains, respectively. Upon cell stimulation, the GEF activity yields Rac2-GTP and the lipase activity yields phosphatidic acid (PA). In the present study, we show for the first time that these activities regulate one another. Upon cell stimulation, both GEF and lipase activities are quickly (within ,3 min) elevated. As soon as it is produced, PA positively feeds back on the GEF and further activates it. Rac2-GTP, on the other hand, is inhibitory to the lipase activity. PLD2 would remain downregulated if it were not for the contribution of the tyrosine kinase Janus kinase 3 (JAK3), which restores lipase action (by phosphorylation at Y415). Conversely, the GEF is inhibited upon phosphorylation by JAK3 and is effectively terminated by this action and by the increasing accumulation of PA at .15 min of cell stimulation. This PA interferes with the ability of the GEF to bind to its substrate (Rac2-GTP). Thus, both temporal inter-regulation and phosphorylation-dependent mechanisms are involved in determining a GEF-lipase switch within the same molecule. Human neutrophils stimulated by interleukin-8 follow a biphasic pattern of GEF and lipase activation that can be explained by such an intramolecular switch. This is the first report of a temporal inter-regulation of two enzymatic activities that reside in the same molecule with profound biological consequences in leukocyte cell migration.

A data-driven network model of primary myelofibrosis: transcriptional and post-transcriptional alterations in CD34+ cells

“…48, 49, 50, 51, 52 PLD2 is dual activity enzyme with both lipase and GEF activities for the Rac2 and RhoA GTPases. As demonstrated by Mahankali et al , 53 in the context of leukocyte chemotaxis, both activities are triggered after cell stimulation, but then, the accumulation of RAC2-GTP, due to GEF action, negatively feeds back on PLD2-lipase activity, and the JAK3 tyrosine kinase restores lipase activity while ultimately inhibiting GEF activity. 54 This lipase-GEF duality has important biochemical and cellular implications and new PLD inhibitors have been recently described.…”

“…54 This lipase-GEF duality has important biochemical and cellular implications and new PLD inhibitors have been recently described. 52, 54 PA is one of the major lipid second messengers that in turn triggers many signaling pathways such as PI3K/Akt/mTOR, sphingosine kinase 1, Raf-1 protein kinase 53 and small GTPases like Ras and Rac. 55 …”

Phospholipase D in Cell Signaling: From a Myriad of Cell Functions to Cancer Growth and Metastasis

“…However, the finding that PA regulates the GEF activity of PLD2 adds a further level of sophistication in the regulation of this enzyme that is necessary considering the key role it has in cellular functions (20). Further, with the discovery of the GEF catalytic site, it is now possible to use lipase-inactive or GEF-inactive mutants to determine lipase or GEF-mediated functions (11).…”

“…Very interestingly, for the dual GEF/lipase activity, the products of the lipase and the GEF reactions regulate the alternate activity. This involves the dual effect of PA on PLD2-GEF activity and a temporal switch in lipase and GEF activities (20).…”

Resolvin D-series regulates Phospholipase D both during inflammation and resolution by modulating phagocyte functions

“…Additionally, PA is conical in shape and carries a negative charge, meaning its accumulation in the membrane results in membrane curvature needed for cell migration (23,(25)(26)(27). Although PLD/PA play a role in macrophage adhesion (25,28), no role of PLD in macrophage polarization has been described as of yet.Phospholipase D (PLD, with the two major mammalian isoforms, PLD1 and PLD2) has a pivotal role in cellular activities such as chemotaxis and phagocytosis (21,(29)(30)(31)(32)(33)(34). Aberrant PLD expression and activity is implicated in inflammation (23,(35)(36)(37)(38) and other cellular functions (21,39).…”