Characterization of the in vivo phosphorylation sites on Syk including a tyrosine in the inter-SH2 domain that modulates the interaction of Syk with the B cell antigen receptor
Abstract
Syk is a non-receptor protein-tyrosine kinase that plays a critical role in the antigen receptor-mediated signaling in B cells. Aggregation of the B cell antigen receptor (BCR) results in recruitment of Syk to the receptor. This association is required for the subsequent activation and tyrosine phosphorylation of Syk. Although both Syk activation and tyrosine phosphorylation are thought to be essential for the activation of downstream signaling pathways, the sites of in vivo phosphorylation have not been reported. To determine sites of in vivo phosphorylation, Myc epitope-tagged Syk was overexpressed in Syk-deficient DT40 chicken B cells and metabolically labeled with ($\sp{32}$P) orthophosphate. Modified tyrosines were identified by comparing the migration pattern of the resulting tryptic phosphopeptides on a 40% polyacrylamide gel with similar maps of previously identified phosphopeptides derived from in vitro autophosphorylated Syk. The results indicate that at least six tyrosines are modified in response to stimulation of B cells. Our results suggest that phosphorylation of a tyrosine (Tyr-130) located between the two SH2 domains causes Syk to dissociate from the antigen receptor. Substitution of this tyrosine with a phenylalanine, which prevents phosphorylation at this site, increased the affinity of Syk for the receptor and enhanced BCR-mediated signaling. In contrast, a glutamate substitution greatly reduced the interaction and resulted in reduced signaling. We also identified another tyrosine (Tyr-317), located in the hinge region between the SH2 domains and the kinase domain, that plays a role in modulating the interaction of Syk with the BCR. Replacement of this tyrosine with a phenylalanine also resulted in increased affinity for the receptor and enhanced signaling. These results reveal a novel mechanism by which Syk function is negatively regulated by factors that modulate its interaction with the receptor.
Degree
Ph.D.
Advisors
Geahlen, Purdue University.
Subject Area
Immunology|Biochemistry|Cellular biology
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