Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor.
Aggregation of the high affinity IgE receptor (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in activation of the protein tyrosine kinases Syk and Lyn. Here, we report that fusion proteins containing the Src homology 2 (SH2) domains of Syk and Lyn precipitated tyrosine-phosphorylated proteins from RBL-2H3 cell lysates. There was no detectable precipitation with the N-terminal Syk SH2 domain, minimal with the C-terminal Syk SH2, and strong when the two SH2 domains were expressed in tandem. The Syk SH2 domains showed pronounced selectivity in the binding of tyrosine-phosphorylated proteins. Thus, Syk SH2 precipitated only three phosphoproteins, two of which were the beta and gamma subunits of Fc epsilon RI. In contrast, the fusion protein with Lyn SH2, in addition to precipitating the same Fc epsilon RI components, bound to many other phosphoproteins. Tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI was essential for their precipitation with the SH2 fusion proteins. By direct binding studies, there was more binding of Syk SH2 to Fc epsilon RI gamma than to Fc epsilon RI beta, whereas Lyn SH2 bound only to Fc epsilon RI beta. The Syk SH2 fusion protein competitively inhibited the association of gamma and beta with Syk in lysates of activated cells. The SH2-mediated association of these two protein tyrosine kinases with Fc epsilon RI could play an important role in receptor signaling.
Kihara
H
H
Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Siraganian
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004792:Enzyme Precursors / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000072377:Syk Kinase; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases
D017931:DNA Primers; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D000072377:Syk Kinase; C000606898:Syk protein, rat; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases