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J. Biol. Chem., Vol. 283, Issue 46, 31348-31355, November 14, 2008

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Sustained Activation of the Tyrosine Kinase Syk by Antigen in Mast Cells Requires Local Ca²⁺ Influx through Ca²⁺ Release-activated Ca²⁺ Channels^*

From the Department of Physiology, Anatomy, and Genetics, University of Oxford, Sherrington Bldg., Parks Road, Oxford OX1 3PT, United Kingdom

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 C1, 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.

Received for publication, June 30, 2008 , and in revised form, September 3, 2008.

^* This work was supported in part by the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A recipient of a GSK-Biotechnology and Biological Sciences and Research Council-Oxford University studentship.

² Held a Christopher Welch Scholarship.

³ To whom correspondence should be addressed. Tel.: 44-1865-272439; Fax: 44-1865-272488; E-mail: anant.parekh{at}dpag.ox.ac.uk.

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