Tyrosine phosphorylation of components of the B-cell antigen receptors following receptor crosslinking.

Abstract

Crosslinking membrane immunoglobulin (mIg), the B-cell antigen receptor, stimulates tyrosine phosphorylation of a number of proteins. Since many receptors are phosphorylated after ligand binding, we asked ifcomponents of the mIg receptor complexes were tyrosine-phosphorylated after mIg crosslinking. Both mIgM and mIgD are noncovalently associated with at least two other proteins. mIgM Is associated with the MB-1 protein, which is disulfide-linked to a protein designated Ig-f3. mIgD is not associated with MB-1 but is with IgD-a, which is also disulfide-linked to Ig-fi. Using immunoprecipitation with a specific anti-MB-i antiserum followed by anti-phosphotyrosine immunoblotting, we found that crosslinking mIgM stimulated tyrosine phosphorylation of (5), were resuspended at 16.7 x 106 cells per ml in a modified Hepes-buffered saline solution (9). Cells were stimulated at 37°C, washed, and resuspended at 108 cells per ml in lysis buffer [20 mM Tris'HCI, pH 8/137 mM NaCl/10% (wt/vol) glycerol/1% Triton X-100/1 mM Na3VO4/2 mM EDTA/1 mM phenylmethylsulfonyl fluoride/20 ,uM leupeptin/0.15 unit of aprotinin per ml] as described (5). Detergent-insoluble material was removed by centrifugation, and protein concentrations were determined by the BCA (bicinchoninic acid) protein assay (Pierce).Anti-MB-i Antiserum. A 34-amino acid peptide from the putative cytoplasmic domain of the murine MB-1 protein [amino acids 187-220 (21) were collected on protein A-Sepharose for 1 hr. The beads were suspended in 0.5 ml of lysis buffer containing 50 ,uM Na3VO4, 0.4% deoxycholate, and 0.3% SDS (wash buffer), layered over 0.4 ml of 30%o sucrose in 0.5 x wash buffer, and centrifuged 5 min at 10,000 x g. The beads were then washed once each with wash buffer and 50 ,uM Na3VO4. Proteins were eluted by boiling in nonreducing SDS/PAGE sample Abbreviations: mlg, membrane immunoglobulin; anti-Tyr(P), antiphosphotyrosine.tTo whom reprint requests should be addressed.tThe first two authors contributed equally to the experiments in this paper. 3436The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Aberrant Trafficking of the B Cell Receptor Ig-αβ Subunit in a B Lymphoma Cell Line

“…Affinity purified goat anti-mouse -chain Abs and goat anti--FITC Abs were obtained from Jackson ImmunoResearch (West Grove, PA). We have previously described the rabbit polyclonal antisera that recognize murine Ig-␣ (28). Abs against murine Ig-␤ were produced by immunizing rabbits with a peptide from the carboxyl-terminal region of Ig-␤ (amino acids 76 -96) (9).…”

“…Protein concentrations were determined using the bicinchoninic acid assay. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the 4G10 anti-phosphotyrosine mAb as previously described (28).…”

Immunoglobulin gamma 2b transgenes inhibit heavy chain gene rearrangement, but cannot promote B cell development.

“…This receptor complex participates in signal transduction events resulting from engagement of mIg (51). In addition, multiple protein tyrosine kinases have been found to associate with mIg and to be activated after crosslinking of the mIg receptor (52)(53)(54) . Many other target substrates havebeen identified downstream from the mIgM receptor (55).…”

Nonredundant Roles of Src-Family Kinases and Syk in the Initiation of B-Cell Antigen Receptor Signaling

“…Engagement of the BCR by Ag leads to the phosphorylation of ITAM in the BCR-associated Iga-and Igb-chains (23). According to the original model of immunoreceptor signaling, this initial ITAM phosphorylation is mediated by SFKs and is a prerequisite for Syk family kinase recruitment and activation (24).…”