Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Amy H. Andreotti
This dissertation structurally examines the intermolecular self-association and quaternary structure of interleukin-2 tyrosine kinase (Itk) and draws conclusions about its relationship to the regulation and signaling of this immunologically important protein. Found primarily in haematopoietic cells, Itk is a member of the Tec family, the second largest family of non-receptor tyrosine kinases. All Tec family members share an SH3, SH2, and catalytic kinase domain structure. The activation of Itk occurs following T-cell receptor response to antigen stimulation. The exact mechanism of regulation in Tec family kinases is unclear. However, intermolecular self-association is emerging as a common characteristic among many members of the Tec family.
To explore the potentially functionally significant self-association in Itk, high-resolution NMR solution structures were solved for the Itk SH3 domain, the Itk SH2 domain, and the Itk SH3/SH2 complex. The non-classical interaction between the Itk SH3 and SH2 domains mediates, in part, the self-association of full length Itk. The structure of the SH3/SH2 complex provides insight on how isomerization of a proline imide bond acts as an intrinsic molecular switch that preorganizes the CD loop of the SH2 domain for a non-classical interaction with the SH3 domain. Additionally, the oligomeric state of Itk self-association is characterized and the SH3/SH2 domain complex is used as a starting point to generate a structural model of the Itk SH3-SH2 fragment self-association that accounts for the oligomerization seen in native gel analysis. The same SH3/SH2 interaction is mutually exclusive with a quaternary structural rearrangement that supports autophosphorylation. Therefore, a structural model is described for Itk autophosphorylation that was generated using a previous point mutational analysis of the Itk SH2 domain coupled with covalent bond restraints found in the linkers of the SH3-SH2-kinase fragment. These studies bring us closer to understanding the structural mechanism behind Itk self-association and describe a model for one of many quaternary structural conformations in which Itk is likely to exist.
Andrew Josef Severin
Severin, Andrew Josef, "Structural characterization of intermolecular self-association in a T-cell specific kinase" (2009). Graduate Theses and Dissertations. 10308.