UC Berkeley

Recognition of phosphotyrosine by SH2 domain-containing proteins is a key feature of signal transduction at the plasma membrane. The output of cell-surface receptors is en- coded in the specificity of these interactions, so understanding the mechanistic factors that determine this specificity is key to determining how information is processed by cells. While the structural details describing how SH2 domains recognize specific sites in their binding partners are well established, much less is known about how the protein context of each SH2 domain and partnership with other recognition modules orchestrate the array of interactions that evolve during a signal transduction event. In theory, combinations of SH2 domain interactions have the ability to provide much higher affinity and specificity than single interactions, but the connection between this property and the molecular mechanisms of protein recognition within known pathways is unclear. To learn how modular protein interactions cooperate to determine specificity, I have studied two differ- ent receptor-binding partner pairs, the ubiquitin ligase c-Cbl with the Epidermal Growth Factor Receptor (EGFR), and the tyrosine kinase ZAP-70 with the T-cell receptor (TCR) I determined that c-Cbl binding to and phosphorylation by EGFR, and the ability of c-Cbl to transfer ubiquitin to the receptor, is sensitive to the oligomeric state EGFR. EGFR dimeri- zation increases the rate of ubiquitin transfer in a manner that does not depend on direct binding by c-Cbl, and this correlates with the requirement for c-Cbl to dimerize in order to become phosphorylated and activated by the EGFR. These findings suggest a way for EGFR ubiquitylation in cells to be fine-tuned by the level of c-Cbl phosphorylation. I have also determined the origins of affinity in the recognition of the T-cell receptor (TCR) by the tyrosine kinase ZAP-70. ZAP-70 uses a tandem SH2 module to bind specific doubly phosphorylated motifs in the TCR with high affinity and specificity. By performing binding experiments and molecular dynamics simulations with tandem SH2 constructs missing specific portions, I determined that the affinity of the tandem SH2 is based SH2 domains with intrinsically low affinity for phosphopeptides that are held together by a linker able to counter electrostatic repulsion between the SH2 domains. In both the c-Cbl-EGFR and ZAP-70-TCR systems, the combination of SH2 domain interactions provides specificity not possible in isolated protein-protein interactions based on single modular domains. The proteins studied here are the central nodes in broad networks of proteins containing mul- tiple modular domains. Understanding how molecular recognition by modular domains supports signal transduction in these systems will require expanding the understanding of specificity to encompass the collective behavior of proteins in cells.