Analysis of tyrosine phosphorylation-dependent interactions between stimulatory effector proteins and the B cell co-receptor CD22

The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphoryl ated on tyrosine in response to cross-linking of the B cell antigen recepto r, thereby generating phosphotyrosine motifs in the cytoplasmic domain whic h recruit intracellular effector proteins that contain Src homology 2 domai ns. By virtue of its interaction with these effector proteins CD22 modulate s signal transduction through the B cell antigen receptor. To define furthe r the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitmen t of the stimulatory effector proteins phospholipase C gamma (PLC gamma), p hosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indica te that the protein tyrosine kinase Syk interacts with multiple CD22-derive d phosphopeptides in both immunoprecipitation and reverse Far Western assay s. In contrast, the Grb2.Sos complex was observed to bind exclusively to th e fourth phosphotyrosine motif ((YENV)-E-828) from CD22 and does so via a d irect interaction based on Far Western and reverse Far Western blotting. Al though both PLC gamma and PI3K were observed to bind to multiple phosphopep tides in precipitation experiments, subsequent studies using reverse Far We stern blot analysis demonstrated that only the carboxyl-terminal phosphopep tide of CD22 ((YVTL)-V-868) binds directly to either one. This finding sugg ests that PLC gamma and PI3K may be recruited to CD22 either through a dire ct interaction with Tyr(868) Or indirectly through an association with one or more intermediate proteins.