Date of Degree
PhD (Doctor of Philosophy)
Jon C.D. Houtman
Despite their essential role in protection, T cells can be dangerous if unregulated. Dysfunctional T cell activity has been implicated in numerous diseases, including the failure of organ transplants, allergic reactions, multiple sclerosis, and coronary artery disease. Signal transduction pathways activated by the T cell receptor (TCR) are good targets for the development of therapies. However, we must first better understand the mechanisms of intracellular signaling that occur when a T cell is activated.
This thesis focuses on the scaffold protein LAT and its role in T cell activation. The localization of signaling proteins into LAT-nucleated complexes and subsequent aggregation of these complexes into microclusters is vital for the activation of intracellular signaling pathways, and the effector functions of T cells. Following TCR stimulation, LAT is phosphorylated and binds SH2 domain containing molecules, such as the adaptor protein Grb2. One LAT molecule is capable of binding up to three Grb2 molecules at one time. Grb2 also binds to the proline rich regions of several proteins, including SOS1. Recent studies indicate that at physiological ratios of Grb2 and SOS1, two Grb2 molecules bind to one SOS1 proline rich region, and this 2:1 stoichiometry is essential for LAT oligomerization and cluster formation.
The interaction of Grb2 and SOS1 is considered to be a model SH3 domain interaction, and has biased our understanding of these relationships for decades. Many studies have focused on the association between the Grb2 SH3 domains and the proline rich region of SOS1. This previous work identified four consensus-binding sites for Grb2 in the proline rich region of SOS1 using short 10-15 amino acid peptides, and indicated that this interaction has a low affinity. Interestingly, the interaction of full SOS1 with Grb2 appears to be at least 100-fold stronger than these peptide-based studies imply. While informative, the use of short peptides leaves the physiological relevance of the peptide-SH3 domain interaction ambiguous.
In this thesis, we specifically emphasize the LAT multi-protein complex and its role in the activation of T cells. First, we compare the differences in the phosphorylation states of various LAT-proximal molecules in two T cell lines and peripheral blood T cells. We then focus on the formation of this complex by investigating the essential interaction between Grb2 and SOS1. Using biochemical and biophysical techniques, we clearly demonstrate that although the previously identified consensus binding sites are important in the context of short peptides, they do not facilitate the interaction of full length Grb2 with the full proline rich region of SOS1. We also attempt to ascertain the role of LAT microclusters in T cell signaling.
xii, 166 pages
Includes bibliographical references (pages 144-166).
Copyright 2012 Rebekah Ruth Bartelt