Ig Beta Research Articles

The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases.

Abstract The B cell Ag receptor complex is comprised of membrane (m)IgM or mIgD noncovalently associated with one or more heterodimers, each containing one subunit of MB-1 (IgM alpha or IgD alpha) and one of B29 (Ig beta or Ig gamma). It is known that cross-linking of the B cell Ag receptor results in protein tyrosine kinase activation. Recent reports from other laboratories have demonstrated that mIg coprecipitates with multiple src family protein tyrosine kinases, including blk, lyn, and fyn. However, the mechanism by which these kinases are physically coupled to the Ag receptor has not been confirmed. It has been hypothesized that the mIg-associated proteins MB-1 and B29 provide a physical link between the Ag receptor (mIg) and one or more protein tyrosine kinases. In this study, we confirm previous findings demonstrating that the B cell Ag receptor coprecipitates with the MB-1/B29 heterodimer as well as the protein tyrosine kinases blk, lyn, and fyn under mild detergent conditions (1% digitonin). Additionally, we demonstrate that in detergent conditions (1% Nonidet P-40 (NP-40)) which disrupt the association between mIg and the MB-1/B29 heterodimer, no protein tyrosine kinase activity can be detected in association with mIg. These findings indicated that NP-40 effectively dissociates the B cell Ag receptor from ancillary signal transducing proteins. MB-1 and B29 were however, found to coprecipitate with blk, lyn, and fyn isolated from B cell lysates containing 1% NP-40. No significant difference was observed in the stoichiometry of association between the kinases and the MB-1/B29 heterodimer in the presence of 1% NP-40 when compared to 1% digitonin. It was further determined that in resting B cells, only a small fraction (approximately 1-3%) of the MB-1/B29 heterodimers appear to be complexed with protein tyrosine kinases. Finally, based on preclearing experiments, it appears that individual heterodimers may associate with a single species of protein tyrosine kinase. These data support the hypothesis that the MB-1/B29 heterodimer couples the antigen receptor to protein tyrosine kinases, thereby providing a physical link that facilitates Ag receptor-mediated regulation of kinase activity.

Characterization of molecular components associated with surface immunoglobulin M in human B lymphocytes: presence of tyrosine and serine/threonine protein kinases.

To characterize the signal transduction through the antigen receptor (AgR) on human B lymphocytes, we analyzed its association with other molecular components. The surface IgM (sIgM) complex isolated in digitonin contains two surface expressed polypeptides--the previously described Ig alpha and Ig beta proteins--covalently linked to each other in a 48/39-kDa heterodimer. We show herein that the human sIgM complex isolated from the Burkitt's lymphoma cell line, Ramos, or from dense tonsillar B cells contains additional molecules--160 kDa and 75 kDa in size--and enzymatic activities able to phosphorylate on tyrosine as well as serine/threonine residues the 39-, 48-, 75- and 160-kDa polypeptides. By specific immunoprecipitation with antibodies to src-family kinases, we consistently detected p56lyn in the sIgM complex. In the Ramos cell line, both p56lck and p59fyn activity were also observed, although to a much lesser extent than p56lyn. These kinases are associated with sIgM before cell stimulation. As shown by two-dimensional electrophoresis, they interact in a tight complex with multimeric forms of the Ig alpha and Ig beta components. The kinases are active in vitro but must be highly regulated in vivo: Western blotting with anti-phosphotyrosine antibodies revealed that stimulation of the AgR on viable B cells increased detectable phosphotyrosine residues on the components present in the sIgM complex. Based on these phosphorylation changes, the 39-, 48-, 75- and 160-kDa molecules are likely to be functionally active elements in an IgM complex crucial for the transduction of the antigenic signal.

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.