Crosstalk between ARF6 and protein kinase Cα in FcγRI-mediated activation of phospholipase D1

Fc receptors play a pivotal role linking the cellular the mechanisms regulating receptor coupling to PLD in intact cells where the components are endogenously ex-and humoral arms of the immune system [1–3]. Our previous studies have shown that the human high-pressed and functional. First, the presence of specific PLD isozymes in the cell membrane fraction was exam-affinity immunoglobulin G receptor Fc␥RI couples to a novel intracellular signaling pathway requiring ined following receptor aggregation over time, using an established protocol [8]. In resting cells, only low levels phospholipase D activation [4]. The mechanisms that regulate receptor coupling to phospholipase D of immunoreactive PLD were found in the nuclear-free membrane fraction. The majority of immunoreactive in intact cells are poorly understood but involve small molecular weight GTPases and protein kinase PLD1 activity was found in the " nuclear " rich fraction which contains the triton-insoluble cytoskeleton (Figure C [5–7]. Here, we show that immune complex aggregation of Fc␥RI stimulates the association of 1a) [9]. Immune complex activation of Fc␥RI resulted in the rapid appearance of immunoreactive PLD1, corre-phospholipase D1 with ARF6 and protein kinase C␣. Surprisingly, PKC␣ activity per se is not required. sponding to the splice variant PLD1a within the nuclear-free membrane fraction (Figure 1a) [10]. No immunoreac-Rather, all of the Fc␥RI-mediated increase in PKC activity requires phospholipase D1, as treatment of tivity for PLD1b or PLD2 was observed in this membrane fraction in resting cells or at any time point after Fc␥RI cells with butan-1-ol (0.3%) or specific downregulation of phospholipase D1 using activation (data not shown). antisense oligonucleotides inhibits Fc␥RI-coupled PKC activation. Moreover, treatment of cells with The regulation of PLD1 activity is complex. Phosphati-butan-1-ol or phospholipase D1 antisense dylinositol-4,5-bis-phosphate is an absolute requirement oligonucleotides inhibits translocation of PKC␦,-⑀, for activity. There is also clear evidence in vitro for the and-␨ but had no effect on the association of PKC␣ regulation of PLD1 by ARF1, Rho, and the regulatory or ARF6 with phospholipase D1. These data indicate domain of either PKC␣ or PKC␤ [5, 11]. However, ex-that association with ARF6 and PKC␣ plays a role tending these observations to intact cells has proved diffi-in coupling Fc␥RI to phospholipase D1 activation cult, as studies have depended on heterologous overex-and that PLD1 lies upstream of all Fc␥RI-mediated pression of various components combined with the use PKC activity. of inhibitors to explore the pathways [6, 12–15]. To determine the role of these …