Abstract
Previous studies have demonstrated that multichain immune recognition receptors, such as the T-cell receptor, signal their occupancy by inducing tyrosine phosphorylation of cellular protein substrates. Type I and II receptors for the Fc portion of IgG are single-chain immune recognition receptors having external, transmembrane, and cytoplasmic domains. In the present study, we have investigated the possibility that, upon engagement, Fc gamma receptors induce protein-tyrosine phosphorylation. Our findings reveal increased phosphorylation of a number of proteins on tyrosine residues after cross-linking of either high (Fc gamma RI) or low (Fc gamma RII) affinity receptors expressed on HL60 cells. Engagement of Fc gamma RII induced rapid tyrosine phosphorylation that decayed to basal levels by 40 min. In contrast, phosphorylation induced by Fc gamma RI cross-linking was more delayed, peaking at 5-10 min, and returning to basal levels by 60 min. Kinase assays of cellular proteins immunoprecipitated from lysates of activated cells by antibody to phosphotyrosine revealed phosphorylation of a 72-kDa molecule that was not present in lysates of resting cells. This phosphoprotein was identified as p72syk by immunoprecipitation with antibodies directed against two different regions of the syk gene product. Immunoprecipitation with antibodies against p72syk followed by immunoblotting with anti-phosphotyrosine antibodies revealed an activation-dependent tyrosine phosphorylation of p72syk. Thus, our present findings demonstrate induction of protein-tyrosine phosphorylation following engagement of monomeric immune recognition receptors and identify p72syk as a tyrosine kinase substrate involved in signaling by Fc gamma RI and Fc gamma RII.
Highlights
Previous studies have demonstratedthat multichain immunerecognition receptors, such as the T-cell receptor, signaltheir occupancy by inducing tyrosine phosphorylation of cellular protein substrates
Recent reports have shown that activation of FcyRI or FcyRII in monocytes and U937 monocytic leukemia cells, respectively, leads to increases in intracellular calcium (Macintyre et al, 1989;Vandewinkel et al, 1990)and tyrosine phosphorylin signaling by FcrRI and FcrRII
FcyRIIA has been shown to induce tyrosine phosphorylation in platelets (Huanget d.,1992). These findings have suggested that tyrosine phosphorylation might play arole in thecoupling of Protein kinases with specificity for tyrosine residues were these receptors to second messenger pathways
Summary
Vol 268,No 21, Issue of July 25, pp. 15900-15905 1993 Printed in 6.S.A. From the Laboratory of CellularDevelopment and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892. Protein-Tyrosine Phosphorylation-HL6O is a promyelomonocytic leukemia cell line that expresses high affinity (FcyRI) as well as low affinity (FcyRII) receptors for IgG. Immunoprecipitation and in Vitro Kinase Assay-Cell extract, prepared from 1 X 10’ cells, was incubated for 1 hon ice with the antibodies indicated and for an additional 30 min with protein ASepharose beads (Pharmacia LKB Biotechnology Inc.) which were, at times, coated with rabbit anti-mouseIgG. Thesefindings strongly suggest that the receptor, not the binding antibody, dictates bins, 1989).Briefly, proteins were fractionated by polyacrylamide gel the time course for tyrosine phosphorylation of cellular subelectrophoresis, transferred onto polyvinylidene difluoride membranes, and digested with 6 N HCl. Hydrolysates were mixed with unlabeled standards and analyzed by thin layer electrophoresis, followed by thin layer chromatography. We conclude that upon activation, both high and low affinity
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