Activation of RBL-2H3 Mast Cells Is Dependent on Tyrosine Phosphorylation of Phospholipase D2 by Fyn and Fgr

Abstract

Both phospholipase D1 (PLD1) and PLD2 regulate degranulation when RBL-2H3 cells are stimulated via the immunoglobulin E receptor, FcRI. However, the activation mechanism for PLD2 is unclear. As reported here, PLD2 but not PLD1 is phosphorylated through the Src kinases, Fyn and Fgr, and this phosphorylation appears to regulate PLD2 activation and degranulation. For example, only hemagglutinin-tagged PLD2 was tyrosine phosphorylated in antigen-stimulated cells that had been made to express HA-PLD1 and HA-PLD2. This phosphorylation was blocked by a Src kinase inhibitor or by small interfering RNAs directed against Fyn and Fgr and was enhanced by overexpression of Fyn and Fgr but not by other Src kinases. The phosphorylation and activity of PLD2 were further enhanced by the tyrosine phosphatase inhibitor, Na 3 VO 4 . Mutation of PLD2 at tyrosines 11, 14, 165, or 470 partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation, although two of these mutations were detrimental to PLD2 function. PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by coexpression of PLD2 and the Src kinases. The findings provide the first description of a mechanism for activation of PLD2 in a physiological setting and of a role for Fgr in FcRI-mediated signaling.

The elucidation of novel SH2 binding sites on PLD2

“…As shown in Figures 5a and b, the F replacement of Y 169 was enough to eliminate PLD activity, whereas Y 179 was dispensable for PLD2 catalysis since mycPLD2 Y179F and DY 179 were fully active enzymes. The same substitution of Y 165 had no effect on PLD2 activity, as described earlier (Choi et al, 2004).…”

“…As for the correlation between PLD2 and tyrosine phosphorylation, it has been previously shown that PLD2 interacts with c-Src and other tyrosine kinases (Slaaby et al, 1998;Ahn et al, 2003;Choi et al, 2004), and the protein tyrosine phosphatase 1b (PTP1b) (Horn et al, 2005). Since the ternary complex PLD2/Grb2/ PTP1b exists in vivo (Horn et al, 2005), it is possible that total tyrosine phosphorylation of PLD2 may also be regulated in a PLD2-Y 179 /Grb2-SH3-dependent manner.…”

“…The PH domain of PLD2 associates with SH2/SH3-containing tyrosine kinases (Ahn et al, 2003;Choi et al, 2004) and the increase in total tyrosine phosphorylation of PLD2 correlates with an apparent increase in PLD activity (Choi et al, 2004). Whether total tyrosine phosphorylation of PLD2 and its high degree of basal activity are physiologically linked is subject to intense investigation (Bourgoin and Grinstein, 1992).…”

The uncovering of a novel regulatory mechanism for PLD2: Formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

“…More recently, others have show that PLD2 can be phosphorylated on tyrosine by Fyn and Fgr kinases in mast cells [24]. This phosphorylation is concomitant with PLD2 activation and cell degranulation [24].…”

Regulation of Rat Basophilic Leukemia-2H3 Mast Cell Secretion by a Constitutive Lyn Kinase Interaction with the High Affinity IgE Receptor (FcεRI)

“…The specific roles of Lyn and Fyn in regulating Fc⑀RI-induced mediator secretion in the RBL-2H3 cell line are still being elucidated. A recent study by Choi et al (22) determined that the Src family kinases Fyn and Fgr positively regulate degranulation in RBL-2H3 cells, at least in part, by activating phospholipase D 2 . In the current study, we stably transfected the unique domain of Lyn into RBL-2H3 mast cells and examined the consequences on Fc⑀RI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion.…”