Abstract
LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.
Highlights
Following engagement of the Fc⑀RI on mast cells, several adaptors, such as SLP-76, Gab2, LAT, and NTAL/LAB, are phosphorylated
LatϪ/Ϫ mast cells have reduced phosphorylation of SLP-76, PLC-␥1, and PLC-␥2, and diminished calcium initiated from the Fc⑀RI; they are still capable of fluxing calcium and undergo relatively normal Erk activation
Disruption of the Lax gene in mice has no impact on lymphocyte development, LaxϪ/Ϫ T and B cells are hyperresponsive and have a slightly enhanced calcium flux, protein tyrosine phosphorylation, and MAPK and Akt activation upon engagement of the T or B cell antigen receptors [20]
Summary
Antibodies—The following antibodies were used for Western blotting: polyclonal and monoclonal anti-LAX [19], monoclonal anti-LAT and LAB [13], anti-pTyr (4G10), the p85 subunit of PI3K, and PLC-␥1 (Upstate Biotechnology); anti-Grb, Erk, Lyn, and Syk (Santa Cruz Biotechnology); anti-pp, p38, pJNK, JNK, pErk, pLyn, pSyk, pAKT(Ser), and Akt (Cell Signaling), biotin-conjugated anti-Fc⑀RI␣, allophycocyanin-conjugated anti-c-Kit, and phycoerythrin-conjugated-Annexin V (eBioscience). Staining and Counting of Mast Cells—Mast cells in the peritoneal cavity, skin, and ear were stained with toluidine blue. Peritoneal cells were spun onto microscope slides by cytospin
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