Consistent with previous studies in other cell types, Fig. 4A demonstrates that both Syk kinase and PI3K are required for phagocytosis in RBL cells [1,

2]. Our data also indicate that FcγRIIA mediated serotonin secretion is dependent on PI3K for full activity. We observed that see more inhibition of PI3K by the inhibitor wortmannin, at the low concentrations that are sufficient to abolish phagocytosis, also reduces FcγRIIA-mediated serotonin secretion by nearly 50% (Fig. 4B). Inhibition of Syk with the Syk-selective inhibitor piceatannol did not significantly inhibit serotonin release by cells expressing WT FcγRIIA, despite the fact that the concentration used (25 μg/ml) had been previously shown to reduce other Syk functions in RBL cells, including serotonin secretion mediated by other Fc receptor isotypes [21, 24]. These data indicate that signaling for FcγRIIA-mediated serotonin release can bypass Syk kinase in RBL cells. In B cells, Syk kinase acts proximal to PI3K [25]. Likewise, in neutrophils stimulated through their IgG receptors, piceatannol treatment blocked the activation of PI3K, indicating that Syk acts proximal to PI3K [25, 26]. Our observation that FcγRIIA-mediated serotonin

release is sensitive to PI3K inhibition but independent of Syk thus appears at odds with a current concept that Syk kinase, recruited early to the phosphorylated ITAM, must serve as an adapter to recruit PI3K for FcγR signaling. Rather, our data suggest that stimulation of FcγRIIA Idasanutlin in vitro may directly engage PI3K and that this event is sufficient to initiate serotonin release. This sequence of events is consistent with other studies that indicate that PI3K can specifically bind to a phosphorylated ITAM without prior involvement of Syk kinase [27]. On the the basis of their discovery that PI3K can bind the phosphorylated ITAM independently of Syk, Cooney et al. proposed a model for phagocytic signaling whereby

Syk and PI3K function in parallel [27]. It is highly possible that their discovery of PI3K’s direct recruitment to the phosphorylated ITAM of FcγRIIA has significant implications for secretion signaling. Our current studies likewise suggest a direct signaling role for PI3K. Since pharmacologic blockade of Syk does not reduce secretion, signaling via Syk appears less involved. While we cannot yet definitively state that there is a direct interaction between FcγRIIA and PI3K, our experiments clearly demonstrate that FcγRIIA-mediated signaling for secretion utilizes ITAM tyrosines and downstream signaling agents different from those required for phagocytosis [3, 27]. These observations are consistent with evidence that the ITAM requirements for FcγRIIA triggered phagocytosis and endocytosis are very different. Specifically, mutations of ITAM tyrosines that completely block phagocytosis do not effect endocytosis.