Degree Type

Dissertation

Date of Award

2014

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Amy Andreotti

Abstract

This thesis dissertation addresses the regulatory mechanism of Tec family kinases at the molecular level, focusing on two members, Interleukin-2 inducible T cell kinase (Itk) and Bruton's tyrosine kinase (Btk). Itk, expressed in T cell lines, modulates the signal relayed from the T cell receptor on the cellular membrane to the nucleus, which results in expression of the genes essential for T cell immunity upon T cell stimulation. Btk is the counterpart of Itk expressed in B cells and is important for the antibody production. Therefore, the activity of Itk and Btk needs to be maintained in a normal range: too low their activity leads to immunodeficiency while too high activity is responsible for certain autoimmune diseases. Better understanding of the molecular details on how the activity of Itk and Btk is regulated will provide new insights for design of small molecules to rescue the misregulated Tec family kinases in these disease states.

Formation of a productive kinase-substrate complex is one prerequisite for efficient catalysis. Generally, protein kinases are quite specific in selecting their substrate, but how the specificity is achieved is less well understood. As for Itk, our lab previously identified a remote docking interaction between the kinase domain of Itk and the C-terminal SH2 domain of PLCγ1, contributing to the recognition of Y783 of PLCγ1 as Itk substrate. In chapter 2, I proceeded on to map the docking interaction surface on Itk kinase domain to the G helix region, using a combination of biochemical and biophysical methods. Elucidation of this substrate recognition surface on Itk provides with an alternative site, other than the kinase active site, that small molecules can target to inhibit Itk activity with the promise of achieving higher specificity.

The active protein kinases share several structural features in common, including the formation of a salt bridge between a conserved lysine on the β3 strand and a conserved glutamate on the αC helix. In the kinase inactive states, this salt bridge breaks apart. Taking advantage of this structural signature, in chapter 3, I proposed a novel method of coupling reductive methylation chemistry and NMR spectroscopy to examine the activation status of protein kinases. I demonstrated using a well characterized protein kinase Src that the conformational transition between the inactive state and the active state of Src can be followed by the spectral change of the peak resonances corresponding to the beta 3 strand lysine and another lysine located on the αC helix. This method can be easily applied to other protein kinases, such as Tec family kinases, regardless of their required expression systems. It complements other structural techniques by providing lysine-specific information on the conformations protein kinases sample in solution. For instance, it can be used to screen and classify small molecules that drive the conformational equilibrium of a particular protein kinase towards the active or the inactive state.

We are also interested in the motifs that regulate the catalytic activity of Tec family kinases. Chapter 4 describes the discovery of a mutation Y617P in the kinase domain of Btk, which not only improves the solubility but also enhances the catalytic activity of Btk kinase domain. The site of the mutation is remote from the active site and therefore how this mutation impacts on the kinase activity is rather intriguing. Coupling biochemical and computational tools, we investigated the known structural features required by an active protein kinase and our data suggested that Y617P activates Btk activity via stabilization of the regulatory spine in the kinase domain. Discovery of the Y617 allosteric site in Btk also raises the possibility of intervening Btk functionality by targeting this site.

In summary, the work presented in this dissertation adds to our knowledge of the molecular mechanism Tec family kinases exploit to attain substrate specificity and catalytic activity. It also provides an experimental tool with potential to facilitate the structure-function study on not only Tec family kinases but also other therapeutically important protein kinases.

DOI

https://doi.org/10.31274/etd-180810-83

Copyright Owner

Qian Xie

Language

en

File Format

application/pdf

File Size

187 pages

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