Differential phosphorylation of functional tyrosines in CD19 modulates B-lymphocyte activation

• Published in: European journal of immunology 2010-04, Vol.40 (4), p.1192-1204
• Main Authors: Ishiura, Nobuko, Nakashima, Hiroko, Watanabe, Rei, Kuwano, Yoshihiro, Adachi, Takahiro, Takahashi, Yoshimasa, Tsubata, Takeshi, Okochi, Hitoshi, Tamaki, Kunihiko, Tedder, Thomas F, Fujimoto, Manabu
• Format: Article
• Language: English
• Subjects: Animals; Antigens, CD19 - chemistry; Antigens, CD19 - genetics; Antigens, CD19 - metabolism; B cell; B-Lymphocytes - immunology; B-Lymphocytes - metabolism; CD40 Antigens - immunology; Cell Line, Tumor; Cell‐surface molecule; Crosses, Genetic; Immunoglobulin G - immunology; Immunoglobulin M - immunology; Lymphocyte Activation; Lymphoma, B-Cell - pathology; Membrane Microdomains - metabolism; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Phosphotyrosine - analysis; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt - metabolism; Proto-Oncogene Proteins c-vav - metabolism; Receptors, Antigen, B-Cell - immunology; Receptors, Antigen, B-Cell - metabolism; RNA, Small Interfering - pharmacology; Signal transduction; Signal Transduction - immunology; Specific Pathogen-Free Organisms
• ISSN: 0014-2980; 1521-4141
• DOI: 10.1002/eji.200939848

CD19 is a B-cell transmembrane molecule that is critical for B-cell activation. CD19 serves as a scaffold protein for key signal transduction molecules including Lyn, PI3K, and Vav, by providing docking sites for these molecules via phosphorylation of CD19-Y⁵¹³, CD19-Y⁴⁸², and CD19-Y³⁹¹. We investigated the process of CD19 tyrosine phophorylation during B-cell activation using Ab specific for each of these phosphorylated tyrosines. BCR engagement induced differential tyrosine phosphorylation, as CD19-Y⁵¹³ phophorylation occurred first, and CD19-Y⁴⁸² phosphorylation was delayed and transient. Different BCR isotypes exhibited distinct patterns of CD19 phosphorylation: IgG-BCR ligation resulted in faster phosphorylation of CD19-Y⁵¹³ and more intense phosphorylation of CD19-Y³⁹¹ than IgM-BCR ligation. This affected CD19-mediated downstream pathways involving Vav, PI3K, and Akt. Additionally, the phosphorylation profile of CD19 differed distinctly according to its plasma membrane location. CD19 phosphorylated at Y⁵¹³ was almost exclusively located within lipid rafts, whereas phosphorylated Y⁴⁸² and Y³⁹¹ were found both inside and outside of the rafts. Furthermore, the phosphorylation of all three tyrosines was remarkably enhanced and prolonged following the simultaneous stimulation of BCR and CD40. Thus, variations in phosphorylation patterns may contribute to the complexity of CD19-regulated signal transduction.