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Molecular and Cellular Biology, March 2006, p. 1826-1838, Vol. 26, No. 5
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.5.1826-1838.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Disruption of SLP-76 Interaction with Gads Inhibits Dynamic Clustering of SLP-76 and Fc{varepsilon}RI Signaling in Mast Cells{dagger}

Michael A. Silverman, Jonathan Shoag, Jennifer Wu, and Gary A. Koretzky*

Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received 18 August 2005/ Returned for modification 19 September 2005/ Accepted 16 December 2005

We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal Fc{varepsilon}RI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for Fc{varepsilon}RI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon Fc{varepsilon}RI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the Fc{varepsilon}RI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits Fc{varepsilon}RI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following Fc{varepsilon}RI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and Fc{varepsilon}RI-induced mast cell responses.

* Corresponding author. Mailing address: University of Pennsylvania School of Medicine, 415 BRBII/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 746-5522. Fax: (215) 746-5525. E-mail: koretzky{at}mail.med.upenn.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, March 2006, p. 1826-1838, Vol. 26, No. 5
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.5.1826-1838.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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