Identification of the Fc epsilonRI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils.
BACKGROUND
In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production.


RESULTS
The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion).


CONCLUSIONS
Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.
University of New Mexico School of Medicine, Department of Pathology, Asthma Research Center, Albuquerque, USA.
C L
CL
Kepley
B S
BS
Wilson
J M
JM
Oliver
D015549:Basophil Degranulation Test; D001491:Basophils / Q000201:enzymology*; D015153:Blotting, Western; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006636:Histamine Release; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017455:Receptors, IgE / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D017455:Receptors, IgE; D013267:Stilbenes; C041525:3,3',4,5'-tetrahydroxystilbene; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; C084944:TYRO3 protein, human; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases