We analyzed the effects of the Janus kinase 3 (Jak3)-specific inhibitor WHI-P131 (4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) and the Jak3/Syk inhibitor WHI-P154 (4-(3′-bromo-4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) on the antigen-induced activation of mast cells. In the rat mast cell line RBL-2H3, both WHI-P131 and WHI-P154 inhibited the antigen-induced degranulation and phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK). The phosphorylation of Gab2, Akt and Vav was also inhibited by WHI-P131 and WHI-P154, indicating that these inhibitors suppress the activation of phosphatidylinositol 3-kinase (PI3K). In bone marrow-derived mast cells (BMMCs) from Jak3-deficient (Jak3−/−) mice, degranulation and activation of MAPKs were induced by the antigen in almost the same extent as in BMMCs from wild-type mice. In addition, the antigen-induced degranulation and activation of MAPKs were inhibited by WHI-P131 and WHI-P154 in both groups of BMMCs, indicating that these compounds inhibit a certain step except for Jak3. The antigen-induced increase in the activity of Fyn, a probable tyrosine kinase of Gab2, was also inhibited by WHI-P131 and WHI-P154 in RBL-2H3 cells. In BMMCs from Jak3−/− mice, the antigen stimulation induced tyrosine phosphorylation of Fyn, which was inhibited by WHI-P131, as well as in BMMCs from wild-type mice and in RBL-2H3 cells. These findings suggest that Jak3 does not play a significant role in the antigen-induced degranulation and phosphorylation of MAPKs, and that WHI-P131 and WHI-P154 inhibit the PI3K pathway by preventing the antigen-induced activation of Fyn, thus inhibiting the antigen-induced degranulation and phosphorylation of MAPKs in mast cells. Keywords: Mast cells, WHI-P131, WHI-P154, p44/42 MAP kinase, p38 MAP kinase, c-Jun N-terminal kinase, phosphatidylinositol 3-kinase, Fyn Introduction Mast cells contribute to inflammatory responses by releasing preformed mediators such as histamine and serine proteases, and generating eicosanoids and cytokines (Williams & Galli, 2000). The early signaling events in antigen-stimulated mast cells are initiated by the tyrosine kinases Lyn and Syk (Beaven & Metzger, 1993; Beaven & Ozawa, 1996). The aggregation of the IgE high-affinity receptor I (FcɛRI) induced by the antigen results in the tyrosine phosphorylation of the β- and γ-chains of FcɛRI by Lyn. The phosphorylation of these chains promotes the recruitment of Lyn and Syk to the β-chain and the γ-chain, respectively, resulting in the tyrosine phosphorylation of linker proteins such as linker for activation of T cells (LAT). Furthermore, the tyrosine kinase Fyn is required for mast cell degranulation (Parravicini et al., 2002). Fyn phosphorylates a linker protein Gab2 (Parravicini et al., 2002), and leads to the activation of phosphatidylinositol 3-kinase (PI3K) (Gu et al., 2001; Wilson et al., 2001). PI3K regulates the translocation of Bruton's tyrosine kinase (Btk) (Buhl & Cambier, 1999; Varnai et al., 1999) to membrane, promoting the activation of Btk by Lyn/Syk (Rawlings et al., 1996; Baba et al., 2001). The activation of these tyrosine kinases induces the tyrosine phosphorylation of phospholipase (PL) Cγ (Li et al., 1992) and Vav (Hirasawa et al., 1995b). The former leads to the generation of inositol 1, 4, 5-trisphosphate and diacylglycerol, which induce an increase in the intracellular Ca2+ level and the activation of protein kinase C, respectively. The latter activates low molecular weight G proteins such as Ras and Rac (Gulbins et al., 1994; Han et al., 1998; Abe et al., 2000), resulting in the activation of the mitogen-activated protein kinase (MAPK) family. The activation of MAPKs in mast cells causes the release of arachidonic acid (Hirasawa et al., 1995a) and the production of cytokines such as interleukin (IL)-4 (Hirasawa et al., 2000) and IL-13 (Hirasawa et al., 2003). Janus kinase 3 (Jak3), a member of the Jak family of cytoplasmic nonreceptor tyrosine kinases, is selectively expressed in hematopoietic cells (Johnston et al., 1994; Witthuhn et al., 1994) and associates with the γc-chain of receptors for IL-2, 4, 7, 9, 15 and 21 (Chen et al., 1997; Asao et al., 2001). Jak3 mediates cytokine-induced responses by activating the cytoplasmic latent forms of signal transducers and activators of transcription (STATs) via phosphorylation of a specific tyrosine residue near the SH2 domain (Leonard & O'Shea, 1998). In addition, Jak3 has been suggested to play important roles in the FcɛRI-mediated activation of mast cells (Malaviya & Uckun, 1999; Malaviya et al., 1999, 2000) and T-cell receptor-mediated activation of T cells (Tomita et al., 2001). In Jak3-deficient (Jak3−/−) mice, the anaphylactic reaction was impaired with defective immune responses (Malaviya & Uckun, 1999; Malaviya et al., 1999). Jak3 also plays roles in bacterial clearance and neutrophil recruitment to the sites of infection by regulating the release of tumor necrosis factor-α from mast cells (Malaviya et al., 2001). In addition, stimulation of the rat mast cell line RBL-2H3 with antigen induced the activation of Jak3 and the specific Jak3 inhibitor WHI-P131 (4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) (Sudbeck et al., 1999) inhibited the antigen-induced degranulation, production of tumor necrosis factor-α and increase in the cytosolic Ca2+ level without affecting the activation of Syk (Malaviya et al., 1999). However, the precise role of Jak3 in the antigen-triggered signaling events in mast cells remains to be clarified. In this study, we evaluated the effects of the specific Jak3 inhibitor WHI-P131 and the Jak3/Syk inhibitor WHI-P154 (4-(3′-bromo-4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) (Ghosh et al., 1999) on the IgE/FcɛRI-mediated activation of RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) from Jak3−/− mice and wild-type mice, and found that these inhibitors strongly suppressed the antigen-induced degranulation and phosphorylation of MAPKs in mast cells via the Jak3-independent pathway.