Quantitative time-resolved phosphoproteomic analysis of mast cell signaling.
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 FcepsilonRI-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 FcepsilonRI 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.
Cao
Lulu
L
Department of Chemistry, Brown University, Providence, RI 02912, USA.
Yu
Kebing
K
Banh
Cindy
C
Nguyen
Vinh
V
Ritz
Anna
A
Raphael
Benjamin J
BJ
Kawakami
Yuko
Y
Kawakami
Toshiaki
T
Salomon
Arthur R
AR
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D013997:Time Factors
D000199:Actins; D010750:Phosphoproteins