The role of ubiquitination in Cbl-mediated negative regulation of the Src-family and Syk/ZAP-70 tyrosine kinases

The intensity of cellular activation in response to antigen receptor engagement determines the level and duration of the immune response. Suboptimal early signals can result in the failure to mount an immune response leading to increased susceptibility to infections. In contrast, too robust an early response can lead to uncontrolled lymphocyte activation and proliferation resulting in host tissue damage and/or autoimmunity. Therefore, understanding the regulatory mechanisms that determine the intensity of early signaling events initiated downstream of antigen receptors is a critical goal in immunology.; Activation of two distinct families of cytoplasmic protein tyrosine kinases, Src-family and Syk/ZAP-70 kinases, is the earliest biochemical response to antigen receptor engagement and is absolutely required for initiating cellular activation. While numerous studies have focused on identifying and characterizing the proteins that relay activation signals downstream of tyrosine kinases, less is known about mechanisms that lead to the downregulation and modulation of tyrosine kinases themselves. One evolutionarily conserved mechanism to regulate enzymatic function is through interactions with negative and positive regulatory proteins. Recent biochemical and genetic studies have identified the proto-oncogene product Cbl as a negative regulator of receptor tyrosine kinases. The following dissertation focuses on characterizing how Cbl regulates the Syk/ZAP-70 family of non-receptor tyrosine kinases and two related but functionally distinct members of Src-family kinases, Fyn and Lck. Specifically, this dissertation (1) identifies and characterizes the Cbl binding site in Syk; (2) demonstrates that Cbl functions as a negative regulator of Syk and ZAP-70 in a manner dependent on an intact tyrosine kinase binding and RING finger domains of Cbl; (3) identifies ubiquitination as the mechanism of this regulation; (4) demonstrates that Cb1 also negatively regulates the Src-family kinases Fyn and (5) Lck by enhancing their degradation via ubiquitination; and (6) characterizes the ubiquitin ligase activity of Cbl as a novel biochemical mechanism to negatively regulate antigen receptor signal transduction.