The long term goal is to define crystallographically the molecular interactions of Cbl in tyrosine kinase signal transduction. Cb1 is the product of the c-cbl proto-oncogene, and is an important element of signaling pathways activated by many growth factor, cytokine, and immune-cell receptors. Cb1 directly binds and inhibits target tyrosine kinases. The proposed structural analysis of Cbl, alone and in complex with the T-cell tyrosine kinase ZAP-70, will reveal mechanisms of recognition and catalytic regulation that are of fundamental importance in tyrosine kinase signaling. Further, these studies will provide the structural foundations for development of novel immunomodulatory and anti-cancer drugs. The structure of an N-terminal fragment of Cbl (Cbl-N) that includes its transforming and phosphotyrosine-binding activities is described in the preliminary studies. Unexpectedly, this region of Cbl is found to include an EF-hand calcium binding domain, a divergent Src homology-2 (SH2) domain, and a four-helix bundle. Calcium fluxes are an essential element of T-cell activation, and Cbl may represent an important but previously unrecognized target of this signal. The structural arrangement of the three domains in Cbl-N suggests that they may cooperate in recognition of phosphoproteins, and that recognition may be regulated by binding of calcium to the EF-hand domain.
The specific aims of this proposal are 1) to test this hypothesis by direct biochemical and structural analysis, and by collaborative structure/function analysis of Cbl's function in T-cell activation and cellular transformation, 2) to determine the structure of Cbl in complex with phosphopeptides derived from target kinases. In Cbl in complex with full-length ZAP-70, in order to see directly how it binds and inhibits target kinases. In addition to the 2.2A resolution structure of Cbl-N, preliminary studies have yielded structure-grade crystals of Cbl-N in complex with a twelve residue ZAP-70 phosphopeptide. A larger fragment of Cbl including the RING-finger domain has been expressed and purified; characterization of this protein suggests that it is suitable for structural studies. Full length ZAP-70 has been expressed and purified using a Baculovirus system; crystallographic analysis of this protein in complex Cbl will synergize with parallel efforts to determine the structure of ZAP-70 bound to the zeta-chain of the T-cell receptor.
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