In Vivo and In Vitro Specificity of Protein Tyrosine Kinases for Immunoglobulin G Receptor (FcγRII) Phosphorylation

Abstract

Human B cells express four immunoglobulin G receptors, FcγRIIa, FcγRIIbl, FcγRIIb2, and FcγRIIc. Coligation of either FcγRIIc isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcγRIIa/c and FcγRIIb1 isoforms become phosphorylated. To identify the FcγRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcγRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcγR isoforms and point mutants. While each PTK phosphorylated FcγRIIa/c, FcγRIIb1 was phosphorylated by Lyn and Blk whereas FcγRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcγRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcγR^– B-cell line transfected with wild-type or mutated FcγRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcγRIIa/c was sufficient to abolish FcγRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcγRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcγRIIa/c and FcγRIIb1 phosphorylation in vivo.