Activation of thymus-derived (T) lymphocytes is critical in the immune response to pathogens, autoimmunity, and in allergic responses. Activation of T cells through their antigen receptors (TCR) involves a complex process of biochemical events critically dependent on protein tyrosine kinases among other signaling effectors. One such tyrosine kinase known as the Inducible T cell kinase (ITK) has been shown to be critical in both T cell development and activation. The complete structure of ITK has not been resolved. However, valuable insights into the structure of ITK have been obtained by NMR analysis of its isolated domains. This analysis predicts two stable conformations of ITK. One is an intramolecular fold resulting from the binding of the SH3 domain to an upstream proline-rich region, whereas the other is a reciprocal dimerization due to the interaction of the SH2 and SH3 domains. The former structure is favored at relatively low concentrations whereas the latter is stable at relatively high concentrations. These predicted structures have not been confirmed in a relevant in vivo system. Therefore, in this application we propose to test the NMR-based predictions of the structure of ITK by generating a panel of biosensor constructs that express chimeric ITK molecules with Cyan Fluorescence Protein (CFP) or Yellow Fluorescence Protein (YFP) at either the amino- or carboxyl-terminus of ITK, or both. These constructs will be expressed in T cells and by using Fluorescence Resonance Energy Transfer (FRET) analysis we will assess whether ITK occurs in an intramolecular fold or an intermolecular dimerization or both. In view of our previous findings that at the resting state ITK is found in the cytoplasm, and upon TCR-induced activation translocates to the T cell-APC contact site, we hypothesize that ITK will be primarily in an intramolecular conformation in the cytoplasm (relatively low concentration) at the resting state, but upon TCR engagement, when ITK translocates to the contact site, intermolecular dimerization (head to head or head to tail) will replace the folded conformation in a time-dependent fashion. Public Health Relevance: Statement Diseases whose pathogenesis has an immunological basis often involve activated lymphocytes that function abnormally. Intracellular molecules known as Tyrosine Kinases are key regulators of lymphocyte activation. The Inducible T cell Kinase (ITK), we propose to study here, represents one of these tyrosine kinases. We propose to study the way ITK behaves inside cells and compare it to information that has been derived from in vitro experiments. Our data will result in better understanding of the relationship between the structure and function of ITK and improve our ability to design ways to control the action of ITK, and other similar molecules, in disease conditions.
Diseases whose pathogenesis has an immunological basis often involve activated lymphocytes that function abnormally. Intracellular molecules known as Tyrosine Kinases are key regulators of lymphocyte activation. The Inducible T cell Kinase (ITK), we propose to study here, represents one of these tyrosine kinases. We propose to study the way ITK behaves inside cells and compare it to information that has been derived from in vitro experiments. Our data will result in better understanding of the relationship between the structure and function of ITK and improve our ability to design ways to control the action of ITK, and other similar molecules, in disease conditions.
| Hirve, Nupura; Levytskyy, Roman M; Rigaud, Stephanie et al. (2012) A conserved motif in the ITK PH-domain is required for phosphoinositide binding and TCR signaling but dispensable for adaptor protein interactions. PLoS One 7:e45158 |
| Grasis, Juris A; Tsoukas, Constantine D (2011) Itk: the rheostat of the T cell response. J Signal Transduct 2011:297868 |
