INTRODUCTION: We have shown that mast cell degranulation is dependent on a rise in intracellular calcium (calcium signal)in concert with activation of protein kinase (PK) C and phospholipase(PL)D. We have focussed our attention on PLD in recent years because least is known about the role of this enzyme. As we and others have shown, PLD is activated in isolated mast cells and a variety of cultured mast cell lines by antigen and other stimulants. The correlations between PLD activation and degranulation under a variety of experimental conditions and the ability to suppress degranulation with primary alcohols, which divert production of the PLD product phosphatidic acid to the corresponding phosphatidyl alcohol, suggest that PLD has a critical role in degranulation (Chadi et al., Mol. Immunol. 38:1269, 2001). Furthermore, we have shown that the two known isoforms of PLD, PLD1 and PLD2, reside in different locations within the cell. PLD1 is associated with Golgi and granule membranes and PLD2 with the plasma membrane (Choi et al., J. Immunol. 278:12039, 2002). Both isoforms regulate different phases of degranulation in mast cells, granule-associated PLD1 in the translocation of granules to the plasma membrane and plasma membrane-associated PLD2 in the fusion of granules with the plasma membrane. However, the mechanisms by which PLD is activated and its downstream targets remain largely undefined.? ? OBJECTIVES: We are pusuing two lines of investigation: the first is to elucidate the mechanisms of activation of PLD and the second to identify downstream events that are regulated by PLD. PLD2 is of particular interest because its location at the plasma membrane makes this isoform accessible to Fc{epsilon}RI-associated Src kinases and other signaling molecules that are recruited by adaptor proteins such as LAT and NTAL within """"""""lipid rafts"""""""" in the plasma membrane (Refs 1 and 2). Last year we reported that PLD2, but not PLD1, associates with and is phosphorylated by the Src kinases, Fyn and Fgr, and that this phosphorylation regulates PLD2 activation and degranulation. For example, phosphorylation PLD2 was blocked by Src kinase inhibitors or by siRNAs directed against Fyn and Fgr and was enhanced by overexpresssion of Fyn and Fgr but not by other Src kinases. Mutation of PLD2 at phosphorylated tyrosines (11, 14, 165, or 470) partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation as well as degranulation (Choi et al., Mol. Cell. Biol. 24:6980, 2004). These earlier studies also revealed that PLD2 co-localized with """"""""lipid rafts"""""""" and that phosphorylation of PLD2 was dependent on integrity of these """"""""lipid rafts"""""""". We now find that PLD itself is required for integrity of """"""""lipid rafts"""""""" and that PLD2 facilitates activation of the tyrosine kinase Syk which plays a critical role in initiating several signaling cascades in mast cells. Further downstream, we have examined the role of PLD in the activation of PKC and phosphatidylinositol 5-kinase because PLD products are potential activators of these enzymes.? ? PLD2 INTERACTS WITH SYK AND REGULATES SYK ACTIVITY AND DOWNSTREAM EVENTS: We found that PLD2 associates with and promotes activation of Syk which, as noted above is a key enzyme in mast cell activation. Antigen stimulation resulted in co-localization of Syk with PLD2 on the plasma membrane as indicated by co-immunoprecipitation and confocal microscopy. This association was dependent on the initial phosphorylation of Syk by Src kinases, but not on PLD2 activity. In vitro, PLD2 interacted via its Phox homology (PX) domain with recombinant Syk to induce phosphorylation and activation of Syk. Both in vivo and in vitro, the interaction of PLD2 with Syk induced additional phosphorylation and and was necessary for the activation of Syk. Furthermmore, overexpression of PLD2 or catalytically inactive PLD2K758R enhanced antigen-induced phosphorylations of Syk and its downnstream targets, the adaptor proteins LAT and SLP-76, while expression of a PLD2 siRNA blocked these phosphorylations. The interaction of PLD2 with Syk also appeared to be necessary for degranulation as indicated by co-transfection of Syk and PLD2 or catalytically inactive PLD2. These findings indicate a dual role for PLD2 as as a signaling molecule. That is, it can act in a catalytic manner to produce biologically active lipids such as phosphatidic acid and diglycerides and as an adaptor molecule to facilitate signal transduction via Syk (Ref. 3).? ? PLD IS ESSENTIAL FOR ACTIVATION OF DIGLYCERIDE-DEPENDENT ISOFORMS OF PKC, PHOSPHATIDYLINOSITOL 5-KINASE, AND DEGRANULATION: The PLD product, phosphatidic acid, is rapidly converted to diacylglyceride by phosphatidate hydrolase. Diglycerides are well established regulators of calcium-dependent and novel isoforms of PKC while phosphatidic acid is known to activate phosphatidylinositol 5-kinase (PI 5-Kinase) in vitro. The significance of the latter activation has yet to be established in vivo. As reported last year, the production of phosphatidic acid by PLD is the primary source of diacylglycerides in mast cells and is essential for sustained activation of diglyceride-dependent isoforms of PKC and degranulation. Although activation of PKC through PLD is sufficient to induce degranulation with pharmacologic stimulants, additional PLD-dependent processes appear to be necessary for antigen-mediated degranulation (Peng and Beaven, J. Immunol. 174:5201, 2005). One such process could be the activation of Syk as described above. ? ? Additional studies with siRNAs against PLD1 and PLD2 have now shown that knock-down of PLD1 and PLD2 resulted not only in deficient activation of PKC but also a reduction in activity of PI 5-kinase. A reduction in activity of PI 5-kinase was also observed in the presence of primary alcohols to suggest that PLD-derived phosphatidic acid was necessary for PI 5-kinase activity in vivo. The studies also suggested that phosphatidic acid was derived primarily from PLD1 and not PLD2 but this is now being verified with siRNAs that are more selective than our original preparations of siRNAs. PI 5-kinase catalyzes the conversion of phosphatidylinositol 4-phosphate (PIP) to phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is the substrate for PLC and an activator of PLD2 in vitro. Therefore our findings point to the possibility that regulation of PIP2 production by PLD1 may in turn regulate PLD2 activity especialy at later stages of degranulation when granules begin to fuse with the plasma membrane. As observed by confocal microscopy, granule-associated PLD1 is incorporated into the plasma membrane during the fusion process and then recycles back into the Golgi and eventually granule membranes over the course of several hours. Such events are under further investigation as are the respective roles of PIP2 and tyrosine phosphorylation of PLD2 in the activation of PLD2.? ? PLD AND LIPID RAFT FUNCTION: Suppression of PLD function, either with primary alcohols or siRNA technology, results in dispersal of lipid raft components including LAT, NTAL, Thy1, and GPI. In antigen stimulated mast cells, the association of the IgE receptor, Fc{epsilon}RI, with lipid raft components is blocked as are downstream phosphorylation events. Consistent results were obtained whether cells were examined by use of fluorescent tagged molecules and confocal microscopy or by classical membrane fractionation techniques (unpublished data). Paradoxically, lipid raft dispersing agents such as methyl beta-cyclodextrin and filipin by themselves caused sustained activation of PLD2.? ? In total, the above results suggest that lipid raft components keep PLD2 quiescent until activation by tyrosine phosphorylation and production of PIP2.
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