Inhibition of Lyn function in mast cell activation by SH3 domain binding peptides.
While Lyn tyrosine kinase has been shown to be necessary for IgE-receptor (FcepsilonRI)-mediated mast cell activation, the mechanism of Lyn activation is not yet understood. Using a micro-electroporation technique to quantitatively introduce peptides into the cytosol of tumor mast cells, we show that proline-rich peptides that preferentially bind Src family SH3 domains block receptor-induced repetitive calcium spikes in a concentration dependent manner. The Src family member Lyn was the likely target, since a series of phage displaying derived peptides with increased Lyn SH3 domain binding specificity inhibited FcepsilonRI-mediated calcium signaling at concentrations consistent with binding to Lyn rather than other Src-type kinases. Furthermore, SH3 binding peptides prevented the plasma membrane translocation of a fluorescently labeled Syk tandem SH2 domain, which binds to phosphorylated FcepsilonRI, suggesting that the peptides specifically block the Lyn-mediated step by which FcepsilonRI cross-linking leads to receptor phosphorylation. Our study suggests that the binding of proline-rich peptides, or corresponding cellular interaction partners, to Lyn SH3 domain suppresses the Lyn-mediated phosphorylatation of FcepsilonRI and calcium signaling.
Stauffer
T P
TP
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, 27710, USA.
Martenson
C H
CH
Rider
J E
JE
Kay
B K
BK
Meyer
T
T
D000595:Amino Acid Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018274:Electroporation; D005456:Fluorescent Dyes; D008407:Mast Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D017455:Receptors, IgE / Q000378:metabolism; D015398:Signal Transduction; D018909:src Homology Domains; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
D005456:Fluorescent Dyes; D010455:Peptides; D017455:Receptors, IgE; D019061:src-Family Kinases; D002118:Calcium