| This dissertation investigates the complex regulation of IL-2 inducible T-cell kinase (Itk) and its related Tec family kinase members, using a combination of experimental and computational approaches. Tec kinases are expressed primarily in hematopoietic cells and regulate key signaling events in lymphocyte activation, differentiation, and development. Itk functions in immune response, fine-tuning signaling downstream of the T-cell Receptor (TCR), and is a putative drug target for allergies and autoimmune disease. Btk, a Tec kinase found in B-cells, carries out a similar role to that of Itk, only downstream of B-cell Receptor (BCR) signaling. The chapters of this dissertation focus on these two kinases, Itk and Btk, and share the common goal of improving our understanding of the molecular mechanisms that regulate these kinases and the immune cell signaling pathways they are a part of, primarily addressing two questions: 1) What is the role of the N-terminal Pleckstrin Homology (PH) domain in regulating Itk and Btk, in addition to its canonical role of PIP3 binding and consequent colocalization to the plasma membrane? 2) The isolated kinase domains of Tec kinases exhibit very weak activity, unlike other kinases domains, such as Src kinases, which have isolated kinases domains that are fully active; what are the sequence/structure differences responsible for rendering Tec kinase domains inactive?;The major contribution of this dissertation is the development of an Itk PH domain mutant, C96E/T110I (referred to later as ItkPHS), that overcomes the insoluble, aggregation-prone nature of wild-type ItkPH without affecting canonical PIP3 binding. This development opened the door to studying the previously intractable Itk PH domain (Chapter 2), and in this dissertation, I elucidate novel functional interactions between ItkPH and Itk kinase domain (Chapter 3), and ItkPH and calmodulin (Chapter 4). Using molecular dynamics simulations, tightly coupled with experiment, I also identify novel N-lobe packing motifs in the kinase domain of the Tec kinase Btk, shedding light on the structural features that account for the weak catalytic activity of the isolated kinase domains these enzymes (Chapter 6). Cumulatively, this work provides new insight into the complex molecular-level regulation of the multi-domain tyrosine kinases in the Tec family. |
