Cross-linking of B cell receptor-related MB-1 molecule induces protein tyrosine phosphorylation in early B lineage...

Cross-linking of surface B cell Ag receptor (BCR) induces tyrosine phosphorylation of BCR-associated components through a receptor-mediated signal transmission pathway. B cell-specific mb-1 and B29 genes encode the alpha/beta components of the BCR-associated complex in mature sIgM+ B cells. Here, we studied the involvement of the mb-1 gene product, MB-1, in the BCR-related structure of immature B cells. Affinity-purified anti-MB-1 antibody coprecipitated mu chain/20-kDa/15-kDa proteins together with monomer MB-1 and Ig-alpha/Ig-beta heterodimer components from digitonin lysates of the pre-B cell line 18.81. The monomer MB-1 and Ig-alpha in the pre-B cell line were shown to migrate with identical patterns in nonequilibrium pH gradient gel electrophoresis/SDS-PAGE. Western blot analysis showed that MB-1 protein is coprecipitated with mu chain from the pre-B cell line. We studied the tyrosine phosphorylation response of bone marrow B lineage cells as well as spleen B cells after cross-linking of BCR-related components with anti-mu, anti-kappa, and anti-MB-1 antibodies. We identified the activation of tyrosine kinase by direct cross-linking of MB-1 expressed on the surface of early B lineage cells. Anti-mu antibody stimulation induced the activation of tyrosine kinase in early (5- to 10-min) and late (30- to 120-min) responses in bone marrow early B lineage cells. Anti-MB-1 mAb (11-18-5) induced the late response exclusively but anti-kappa antibody induced only the early response. These results clearly indicate that MB-1 acts in the BCR-mediated signal transmission in early B lineage cells. To explore the molecular mechanism of protein tyrosine phosphorylation in bone marrow B lineage cells, we studied associated components of the BCR complex by using an in vitro kinase reaction and observed the phosphorylation of a 60-kDa protein in pre-B cell lines. The 60-kDa phosphoprotein coprecipitated with MB-1 and the BCR-related complex is very similar to the Src-type Fyn tyrosine kinase or a Fyn-related protein.