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* Department of Chemistry,
Department of Molecular Biology, Cell Biology, and Biochemistry, and
Department of Computer Science, Brown University, Providence, RI 02912; and
La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037
Mast cells play a central role in type I hypersensitivity reactions and allergic disorders such as anaphylaxis and asthma. Activation of mast cells, through a cascade of phosphorylation events, leads to the release of mediators of the early phase allergic response. Understanding the molecular architecture underlying mast cell signaling may provide possibilities for therapeutic intervention in asthma and other allergic diseases. Although many details of mast cell signaling have been described previously, a systematic, quantitative analysis of the global tyrosine phosphorylation events that are triggered by activation of the mast cell receptor is lacking. In many cases, the involvement of particular proteins in mast cell signaling has been established generally, but the precise molecular mechanism of the interaction between known signaling proteins often mediated through phosphorylation is still obscure. Using recently advanced methodologies in mass spectrometry, including automation of phosphopeptide enrichments and detection, we have now substantially characterized, with temporal resolution as short as 10 s, the sites and levels of tyrosine phosphorylation across 10 min of Fc
RI-induced mast cell activation. These results reveal a far more extensive array of tyrosine phosphorylation events than previously known, including novel phosphorylation sites on canonical mast cell signaling molecules, as well as unexpected pathway components downstream of FcRI activation. Furthermore, our results, for the first time in mast cells, reveal the sequence of phosphorylation events for 171 modification sites across 121 proteins in the MCP5 mouse mast cell line and 179 modification sites on 117 proteins in mouse bone marrow-derived mast cells.
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 This work was supported by National Institutes of Health Grant 2P20RR015578 and by a Beckman Young Investigator Award. B.J.R. is supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.
2 Address correspondence and reprint requests to Dr. Arthur R. Salomon, Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Box G-E335, Providence, RI 02903. E-mail address: art{at}drsalomon.com
3 Abbreviations used in this paper: SH2, Src homology 2; BMMC, bone marrow-derived mast cell; Btk, Bruton’s tyrosine kinase; MS, mass spectrometry; FTMS, Fourier transform MS; Gab2, Grb2-associated binding protein 2; GAP, GTPase-activating protein; Grb2, growth factor receptor-bound protein 2; IMAC, immobilized metal affinity chromatography; ITMS, ion trap MS; LAT, linker for activation of T cells; LC-MS, liquid chromatography MS; MAWD, MAPK activator with WD repeats; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PLC
, phospholipase C; SHC, SH2 domain-containing transforming protein C; SHP 1/2, SH2-containing protein tyrosine phosphatase 1/2; SIC, selected ion chromatogram; SLP76, SH2 domain-containing leukocyte protein of 65 kDa; Sos, son of sevenless homologue; WASP, Wiskott-Aldrich syndrome protein.
4 The online version of this article contains supplemental material.
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