Degree Type

Dissertation

Date of Award

2009

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biophysics

First Advisor

Amy H. Andreotti

Abstract

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.

Copyright Owner

Andrew Josef Severin

Language

en

Date Available

2012-04-28

File Format

application/pdf

File Size

166 pages

Included in

Biophysics Commons

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