Proline cis/trans isomerization regulates a T cell specific tyrosine kinase

This dissertation examines the role of protein-protein interactions that regulate the conformation and function of the Tec family kinase, interleukin-2 tyrosine kinase (Itk). Tec family kinases are expressed in hematopoetic cells and modulate intracellular signaling cascades in response to external stimuli. The domain structure of Tec family members contains the conserved SH3, SH2, and catalytic domains common to many kinase families, yet they are distinguishable by the presence of a unique N-terminal sequence. The mechanism by which Itk is regulated is not well understood. Both nuclear magnetic resonance spectroscopy and functional assays were used to elucidate a novel regulatory mechanism for Itk in the work presented in this dissertation. These studies demonstrate that the Itk SH2 domain adopts two distinct conformations in solution that are in slow exchange. The observed conformational heterogeneity is due to proline cis/trans isomerization around a single prolyl imide bond, generating a cis proline-containing conformer and a trans proline-containing conformer. Each conformer displays unique ligand binding properties. The trans conformer preferentially binds phosphotyrosine-containing ligands, whereas the cis conformer is required for a novel intermolecular interaction with the Itk SH3 domain. This SH3-SH2 self-association interaction is mediated by the conserved aromatic ligand binding pocket on the Itk SH3 domain and a newly defined surface on the Itk SH2 domain.;Proline cis/trans isomerization is not only an important switch in regulating ligand binding, we also observe that the conformationally heterogenous proline residue is required for recognition of Itk as a substrate for the peptidyl-prolyl isomerase, cyclophilin A. Cyclophilin A accelerates the interconversion between the cis and trans Itk SH2 domain conformers. Furthermore, both in vitro and in vivo cellular experiments reveal that cyclophilin A inhibits Itk kinase activity. This observation allows us to propose a mechanistic model for a completely new mode of kinase regulation and reveals a cellular role for cyclophilin A in T cell signaling. In sum, this dissertation provides molecular details about the structure of the Tec family kinase, Itk, and the functional implications of these results