Our past studies have demonstrated that mast cell degranulation is dependent on mobilization of calcium via phospholipase (PL) C and activation of protein kinase (PK) C along with PLD. In recent years we have focussed primarily on on PLD. This enzyme is the primary source of phosphatidic acid and diglycerides, both stimulants of PKC, and PLD activation is essential for degranulation. For example, we have shown that degranulation was suppressed by low concentrations of primary alcohols (but not by secondary and tertiary alcohols)that divert production of the PLD product phosphatidic acid to phosphatidylalcohol, a strategy that is widely utilized to study PLD function because there are no known pharmacologic PLD inhibitors. Also, expression of catalytically inactive forms of each of the two known forms of PLD, PLD1 and PLD2, both of which are present in mast cells, block degranulation (see previous reports in this series). We now show that both PLD1 and PLD2 respond to secretory stimuli and participate in the degranulation process; PLD1 in the translocation of granules and PLD2 in the final fusion of granules with plasma membrane. For example, expression of wild type and catalytically inactive mutants of PLD1 and 2 fused with enhanced green fluorescent protein (EGFP) indicated that PLD1 and PLD2 are located on secretory granules and the plasma membrane, respectively. Both isoforms are activated by antigen via the IgE receptor (Fc-epsilon-RI) and other stimulants. On stimulation, PLD1 migrates along with granules to the cell periphery and fuses with the plasma membrane. Expression of catalytically inactive forms of PLD1 and PLD2, which distribute exactly as their wild type counterparts, blocks migration of granules and their fusion with the plasma membrane, respectively. 1-Butanol but not tertiary butanol also blocks migration and fusion PLD1-labelled granules. These results imply that the activation of the PLDs must be coordinated to achieve completion of both processes of the exocytotic process, i.e. migration and fusion of granules. Our assumption from the above studies was that PLD2 was activated first because of its association with the plasma membrane makes it most accessible to FcepsilonRI associated Src kinases. Kinetc studies suggest this to be the case. In intact cells, PLD in total is activated by antigen and synergistically by stimulants of CaM kinase II, PKC and PKA. However, the activation of PLD by antigen is only partially blocked by inhibitors of these kinases to suggest additional cryptic activation mechanism(s). We have now identified a major cryptic signaling mechanism as follows. Studies with pharmacologic inhibitors revealed that activation of PLD2 is regulated, directly or indirectly, by Src kinases following stimulation of the RBL-2H3 mast cell line by antigen. Further studies revealed that PLD2 but not PLD1 is phosphorylated directly by the Src kinases Fyn and Fgr and that this phosphorylation is essential for PLD activation. For example, following expression of hemagglutinin (HA) -tagged PLDs only HA-PLD2 was tyrosine phosphorylated in antigen-stimulated cells. The phosphorylation of HA-PLD2 was blocked by a Src kinase inhibitor but not by inhibitors of Syk and other protein kinases. Of the Src kinases cloned from and overexpressed in RBL-2H3 cells, only Fyn and Fgr interacted with PLD2 and enhanced antigen-induced PLD2 tyrosine phosphorylation. Mutational studies indicated that PLD2 was phosphorylated at tyrosines- 11, 14, 165, and 470. Mutation of any one site partially impaired PLD2 activation and mutation of all sites blocked activation. These findings have probable biological relevance in that PLD2 phosphorylation precedes degranulation, both events are equally sensitive to inhibition of Src kinase activity, and both are enhanced by co-expression 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 Fc-epsilon-RI-mediated signaling.These studies not only reaffirm an essential requirement of PLD for secretion but also provide the first demonstration of the regulation of PLD2 by a tyrosine kinase and the first defined mechanism of activation of this PLD. Prior to these studies, the mechanism of activation of PLD2 was unclear. PLD1, in contrast is activated through several protein kinases including CaM kinase II and PKC. Our working assumption is that PLD2 is regulated through the Src kinases and PLD1 through activation of PKC and other protein kinases downstream of PLC . As mentioned above, PLD1 is activated by PKC but the presumption that PLD may in turn activate PKC through generation of phosphatidic acid and diglycerides generated from phosphatidic acid is a matter of dispute. However, we find that antigen induced translocation and phosphorylation of various isoforms of PKC is blocked by 1-butanol but not by tertiary butanol as is degranulation. As a control, direct activation of the PKC isoforms by phorbol ester is not affected by the butanols (see previous report in this series). Examination of the different isoforms of PKC have shown that activation of all calcium-dependent and novel isoforms of PKC are blocked by 1-butanol whereas the diglyceride-independent forms of PKC are unaffected. Also, kinases that regulate PKC such as PDK1 are unaffected by 1-butanol. It would appear that production of diglycerides via PLD is indeed essential for PKC activation. We suggest that that PLD and PKC, each of which can activate the other, provide an amplifying loop in the secretory pathway and accounts for the widely reported synergistic interactions of pharmacologic stimulants on mast cell secretion. Studies are now underway utilizing the the siRNA approach to verify these conclusions. Finally, studies of signalling mechanisms in human mast cells and of the inhibitory actions of glucorticoids (discussed in full in another report, see HL000990-17 LMI)lead us to question current assumptions of the early signalling events that result in degranulation and other functional responses of mast cells to antigen. Our studies indicate that PLC-gamma-2 and not PLC-gamma-1 is the stronger contributor in regulating the calcium signal and degranulation. In addition PLC-gamma-2 and not PLC-gamma-1 is dependent on phosphatidylinositol 3-kinase.
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