Abstract
Engagement of many cell surface receptors results in tyrosine phosphorylation of an overlapping set of protein substrates. Some proteins, such as the adaptor protein Shc, and a frequently observed Shc-associated protein, p145, are common substrates in a variety of receptor signaling pathways and are thus of special interest. Tyrosine-phosphorylated Shc and p145 coprecipitated with anti-Shc antibodies following B cell antigen receptor (BCR) cross-linking or interleukin-4 (IL-4) receptor activation in B cells, and after lipopolysaccharide (LPS) treatment or IgG Fc receptor (Fc gamma R) cross-linking in macrophages. In the case of BCR stimulation, we have shown that this represented the formation of an inducible complex. Furthermore, in response to LPS activation or Fc gamma R cross-linking of macrophages and BCR cross-linking (but not IL-4 treatment) of B cells, we observed a similar tyrosine-phosphorylated p145 protein associated with the tyrosine kinase Syk. We did not detect any Shc associated with Syk, indicating that a trimolecular complex of Shc, Syk, and p145 was not formed in significant amounts. By several criteria, the Syk-associated p145 was very likely the same protein as the previously identified Shc-associated p145. The Syk-associated p145 and the Shc-associated p145 exhibited identical mobility by SDS-polyacrylamide gel electrophoresis and identical patterns of induced tyrosine phosphorylation. The p145 protein that coprecipitated with either Shc or Syk bound to a GST-Shc fusion protein. In addition, a monoclonal antibody developed against Shc-associated p145 also immunoblotted the Syk-associated p145. The observations that p145 associated with both Shc and Syk proteins, in response to stimulation of a variety of receptors, suggest that it plays an important role in coordinating early signaling events.
Highlights
Receptors for antigens, cytokines, and growth factors utilize tyrosine phosphorylation of proteins to initiate and propagate intracellular events that result in cellular responses
The Formation of the Shc1⁄7p145 Complex and Its Persistence in the Cell Were Correlated with Tyrosine Phosphorylation—We examined the kinetics of the Shc1⁄7p145 complex formation to determine whether this was a transient response or a prolonged signaling event following B cell antigen receptor (BCR) stimulation (Fig. 1)
As Shc has two domains capable of interacting with other tyrosine-phosphorylated proteins, it is likely that BCR stimulation induced the tyrosine phosphorylation of p145 and this in turn led to Shc binding
Summary
Receptors for antigens, cytokines, and growth factors utilize tyrosine phosphorylation of proteins to initiate and propagate intracellular events that result in cellular responses. As many of these receptors lack intrinsic tyrosine kinase activity, this. Phosphorylation at Tyr-317 of the central domain of Shc directs binding of the Grb-21⁄7SOS-1 complex to Shc via the SH2 domain of Grb-2 [16] By virtue of this association, Shc has been implicated in Ras activation.
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