In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.
Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-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 FcgammaRII 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 FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/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 FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/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 FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.
Bewarder
N
N
Biochemie II, Fakultät für Chemie, Universität Bielefeld, Germany.
Weinrich
V
V
Budde
P
P
Hartmann
D
D
Flaswinkel
H
H
Reth
M
M
Frey
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D007152:Immunologic Capping; D047908:Intracellular Signaling Peptides and Proteins; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D017452:Receptors, IgG / Q000145:classification / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D000072377:Syk Kinase; D014407:Tumor Cells, Cultured; D019061:src-Family Kinases / Q000378:metabolism
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011947:Receptors, Antigen, B-Cell; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; C082599:protein-tyrosine kinase p55(blk); D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases