IN-VIVO AND IN-VITRO SPECIFICITY OF PROTEIN-TYROSINE KINASES FOR IMMUNOGLOBULIN-G RECEPTOR (FC-GAMMA-RII) PHOSPHORYLATION

Human B cells express four immunoglobulin G receptors, Fc gamma RIIa, Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RIIc. Coligation of eithe r Fc gamma RII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the Fc gamma RIIa/e a nd Fc gamma RIIb1 isoforms become phosphorylated. To identify the Fc g amma RII-phosphorylating protein tyrosine kinase (PTK), we used the co mbination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different Fc gamma RII iso forms as well as tyrosine exchange mutants, we show that each of the B CR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphoryl ation patterns with regard to the different Fc gamma R isoforms and po int mutants. While each PTK phosphorylated Fc gamma RIIa/c, Fc gamma R IIb1 was phosphorylated by Lyn and Blk whereas Fc gamma RIIb2 became p hosphorylated only by Blk Mutants lacking both tyrosine residues of th e immune receptor tyrosine-based activation motif (ITAM) of Fc gamma R IIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosp horylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an Fc gamma R(-) B-cell li ne transfected with wild-type or mutated Fc gamma RIIa demonstrated th at exchange of the C-terminal tyrosine of the ITAN of Fc gamma RIIa/c was sufficient to abolish Fc gamma RIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to Fc gamma RIIa/c, with the ITAM being necessary for association. Comparison of the phosp horylation pattern of each PTK observed in vitro with the phosphorylat ion pattern observed in vivo suggests that Lyn is the most likely cand idate for Fc gamma RIIa/c and Fc gamma RIIb1 phosphorylation in vivo.