title: A system for reconstructing B cell antigen receptor signaling in the mouse myeloma J558L cell line.
abstract: B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca(2+) indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lμv11 cells) permitted monitoring of Ca(2+) mobilization. Reconstituting CD19 in J558Lμv11 cells resulted in detectable BCR-induced Ca(2+) mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lμv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling.
authors: Department of Cell signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors: Satoru
authors: S
authors: Harumiya
authors: Aya
authors: A
authors: Yoshino
authors: Koji
authors: K
authors: Hayashizaki
authors: Kazuya
authors: K
authors: Mizuno
authors: Hidetaka
authors: H
authors: Yakura
authors: Takahiro
authors: T
authors: Adachi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018941:Antigens, CD19 / Q000378:metabolism; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D015536:Down-Regulation; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000473:pathology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018941:Antigens, CD19; D010750:Phosphoproteins; D011947:Receptors, Antigen, B-Cell; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D017493:Leukocyte Common Antigens