Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels.
Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.
Ng
Siaw Wei
SW
Department of Physiology, Anatomy, and Genetics, University of Oxford, Sherrington Bldg., Parks Road, Oxford OX1 3PT, United Kingdom.
di Capite
Joseph
J
Singaravelu
Karthika
K
Parekh
Anant B
AB
D000818:Animals; D000941:Antigens / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D015536:Down-Regulation; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008407:Mast Cells / Q000378:metabolism*; D000071740:ORAI1 Protein; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology / Q000378:metabolism; D000072377:Syk Kinase
D000941:Antigens; D015220:Calcium Channels; D047908:Intracellular Signaling Peptides and Proteins; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat; D002118:Calcium