title: Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels.
abstract: 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.
authors.affiliation: Department of Physiology, Anatomy, and Genetics, University of Oxford, Sherrington Bldg., Parks Road, Oxford OX1 3PT, United Kingdom.
authors.forename: Siaw Wei
authors.initials: SW
authors.lastname: Ng
authors.forename: Joseph
authors.initials: J
authors.lastname: di Capite
authors.forename: Karthika
authors.initials: K
authors.lastname: Singaravelu
authors.forename: Anant B
authors.initials: AB
authors.lastname: Parekh
mesh_terms: 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
chemical_list: 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