Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Cbl is an adaptor protein that functions as a negative regulator of many si gnalling pathways that start from receptors at the cell surface(1-4). The e volutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosp horylated tyrosine residues and has cell-transforming activity. Point mutat ions in Cbl, that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential(5). In T cells, Cbl-N binds to the tyrosine-phosphoryla ted inhibitory site of the protein tyrosine kinase ZAP-70(6). Here we descr ibe the crystal structure of Cbl-N, both alone and in complex with a phosph opeptide that represents its binding site in ZAP-70. The structures show th at Cbl-N is composed of three interacting domains: a four-helix bundle (4H) , an EF-hand(7) calcium-binding domain, and a divergent SH2 domain(8) that was not recognizable from the amino-acid sequence of the protein. The calci um-hound EF hand wedges between the 4H and SH2 domains and roughly determin es their relative orientation. In the ligand-occupied structure, the 4H dom ain packs against the SH2 domain and completes its phosphotyrosine-recognit ion pocket. Disruption of this binding to ZAP-70 as a result of structure-b ased mutations in the 4H, EF-hand and SH2 domains confirms that the three d omains together form an integrated phosphoprotein-recognition module.