Visualization of Syk-antigen receptor interactions using green fluorescentprotein: Differential roles for Syk and Lyn in the regulation of receptor capping and internalization

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimula tion of multiple intracellular signal transduction cascades via receptor-as sociated, protein tyrosine kinases of both the Src and Syk families. To mon itor changes in the subcellular distribution of Syk in B cells responding t o BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion pr otein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from c ytoplasmic and nuclear compartments to the site of the cross-linked recepto r at the plasma membrane. The Syk-receptor complexes aggregate into membran e patches that redistribute to form a cap at one pole of the cell. Syk is n ot demonstrably associated with the internalized receptor. Catalytically ac tive Syk promotes and stabilizes the formation of tightly capped BCR comple xes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of th e clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation o f the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating r eceptor internalization and down-regulation.